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The phytochemical characterisation has been considered crucial in the investigation of the invasive potential of alien plant species, making it a primary contributor to their negative impact on the environment [1]. Conversely, the diverse array of phytochemicals also presents an immense untapped potential in the extraction of active compounds, leading to their application as phytopharmaceuticals [2, 3]. This study provides the LC-MS phytochemical screening of four invasive alien plant species (IAPS) (Robinia pseudoacacia L., Helianthus tuberosus L., Ailanthus altissima (Mill.) Swingle, and Solidago canadensis L.) in the Istria region (Croatia). Using a non-targeted metabolomics approach, we identified 130 phenolic compounds in the 70% ethanolic leaf extracts, of which 85 were species-specific. Caffeoylquinic, coumaroylquinic, and feruloylquinic acids, as well as kaempferol and caffeic acid hexosides, were detected in all the tested IAPS. Furthermore, A. altissima had the highest spectrophotometrically determined total phenolic (TP) and non-flavonoid (TNF) content, as well as antioxidant capacity (AC) obtained by ABTS, DPPH, and FRAP assays. The highest total flavonoid (TF) content was recorded in the extracts of H. tuberosus. A strong correlation between TP content and AC values was observed in all extracts. In the next phase, we will investigate the antimicrobial, cytotoxic, genotoxic and antioxidant properties of extracts. Based on the obtained results and the ecological assessment of IAPS impacts, we will propose a model for exploring new IAPS provisioning ecosystem services on the pilot territory of Istria.

Climate change is a global challenge that has changed the physiological and biochemical parameters of plants. Plants are sessile organisms and their survival depends on the effective activation of metabolic mechanisms of resistance to such stress. In the scope of this work, we investigated the influence of low (0⁰C) and high (80⁰C) water temperature on the metabolism of young broccoli plants (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.) with the aim of adefining the parameters that indicate susceptibility or resistance of broccoli to stress caused by low and high water temperature, and b) evaluating the changes in selected parameters of broccoli metabolism due to these two types of stress. For the determination of bioactive compounds, we used high performance liquid chromatography and spectrophotometric methods. For all pigments, except chlorophyll a, a statistically significant difference was observed between the group treated with high temperature water and the remaining two groups (control and group treated with cold water). The highest proportion of chlorophyll b was found in the group treated with hot water. Regarding the proportions of total porphyrins and lycopene, a difference was also observed between the group treated with hot water and the other two groups. The proportion of carotenoids, on the other hand, was the lowest in the group treated with hot water. The proportion of total proanthocyanidins in young broccoli plants was the highest in the group treated with hot water, while the proportion of total hydroxycinnamic acids and flavonols was significantly lower in the group treated with hot water than in the other two groups. The proportion of the remaining four groups of compounds (total phenols, flavonoids, tannins and phenolic acids) in broccoli subjected to stress caused by high and low temperature water did not statistically differ from the control group.

Plants produce specialized metabolites, phenolics being one of them, in order to adapt to abiotic and biotic stresses more successfully. This study aimed to investigate which groups of phenolics in broccoli seedlings (Brassica oleracea botrytis var. cymosa) are more susceptible to concentration changes at high and low growing temperature. To answer this, in the fall of 2022/2023, we cultivated three biological replicas of broccoli seedlings in three different temperature conditions: high (38 °C day/33 °C night), low (12 °C day/7 °C night), and regular (23 °C day/18 °C night) as a control group. Seedlings were lyophilized, extracts in 70% ethanol prepared and spectrophotometric analyses of total phenolics, flavonoids, flavonols, hydroxycinnamic and phenolic acids were conducted. According to the results, total flavonols and flavonoids decreased significantly when seedlings were grown at both high and low temperatures, while total phenolics, phenolic and hydroxycinnamic acids decreased significantly only when grown at high temperature. Out of these groups of phenolics, total flavonols were the most susceptible to change when grown at high temperature, while total flavonoids were the most susceptible to change when grown at low temperature. Total phenolic acids were the most resistant at high and low temperature cultivation. In conclusion, high temperature cultivation significantly reduced the concentration of all analyzed groups of phenolics and had more impact on phenolics of broccoli seedlings than low temperature cultivation.

Plants are extremely adaptable organisms, they can survive different types of environmental stresses. Survival is possible due to their ability to quickly adapt phyotchemically to new conditions. Such phytochemical adaptations can cause change of plant nutritional potential, as well as to lead to formation of new (sub)species. The effect of temperature stress on broccoli plants will be discussed, accompanied by the most important results of analyses.

Due to climate change, sudden and intense changes in weather conditions are becoming more frequent. Immobile organisms such as plants cannot “take refuge” from high or low temperatures but adapt their physiology to the new conditions to survive. The intensity and direction of these changes depend on both the temperature and the plant species. Since adaptations at the metabolic level are fast, changes in the ambient temperature can be detected on the level of biochemical responses by which plants react to the new conditions. Such changes are usually crucial yield-limiting factors for plants. Broccoli (Brassica oleracea) is rich in vitamins (C, K) ; β-carotene, a precursor of vitamin A ; dietary fibers ; polyphenols ; fatty acids ; minerals and glucosinolates - phytochemicals that are predominantly represented in Brassica vegetables. These compounds contribute to the health benefits of broccoli, such as antioxidant, antiproliferative and antidiabetic properties and the protection of the cardiovascular system. Temperature and irradiation have been recognized as the most important factors for the production of consumer-orientated quality broccoli. Since broccoli is native to moderate climatic zones such as the Mediterranean region, high or low temperatures will cause perturbations in its phytochemical profile to survive. Such phytochemical perturbations might have consequences on the biological effects of a plant and its products. In scope of our work, we investigated the influence of hot- and cold-water stress on the metabolism of young broccoli (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.) plants with the aim to (i) define the susceptible and resistant parameters of this plant during low- and high-temperature water stress, (ii) determine the degree of metabolism change of broccoli due to these two types of stress, and (iii) determine the degree of change in the biological effects of broccoli extracts due to the types of stress.

In the last years there is a growing demand for fresh and healthy food, and sprouts are an example of recognized nutritional value. Moreover, the improvement of the foods nutritional quality has become a general health concern. The practical advantage and convenience that the sprouts offer to the consumers makes this market currently the fastest growing sector in food industry. One simple strategy to improve the nutritional properties of microgreens could be the interspecific transfer of bioactive compounds. Therefore, our goal was to analyze the potential of interspecific source-sink phytochemical transfer for the improvement of phenolic bioactive compounds and vitamin C profile in microgreens, their gastrointestinal bioaccessibility, hypoglycemic potential, and antioxidant capacity as well as antiproliferative effect. In addition, we also tested whether an effect of phytochemical transfer is evident in the expression of marker genes in phenolic biosynthetic pathways. Chinese cabbage (Brassica rapa ssp. pekinensis) was used as a model organism. The results showed that interspecific phytochemical transfer from donor Tamus communis extract significantly improved bioaccessibility of vitamin C, kaempferol and total phenolics both before and after gastrointestinal digestion of Chinese cabbage microgreens, increased their hypoglycemic activity, and antioxidant capacity. In addition, sprouts treated with extracts from Rosa sp. and H. perforatum showed better cytotoxicity towards human breast adenocarcinoma cells than untreated sprouts. qRT-PCR analyses revealed that BrpFLS of Chinese cabbage microgreens was slightly upregulated upon a 4-hour treatement with Rosa sp. extract, therefore, this gene could be used as a marker for the biosynthetic pathway alteration. All together, the results suggest that treatment with donor extracts can be considered a promising approach to improve the nutritional properties of Chinese cabbage sprouts as „healthy food“.

Due to inability to culture phytoplasmas in vitro in a pure culture, our knowledge of them is still limited. Nevertheless, their negative impact on agricultural production worldwide is prominent. Phytoplasmas are bacterial parasites which belong to the genus ‘Candidatus Phytoplasma’ and infect a wide variety of plants and insects. Among infecting other agriculturally important plant species, they are known to heavily affect viticultural production. Phytoplasmas have a variety of effector molecules which help them modify host physiological processes in order to promote their reproduction and transmission. However, plant responses to phytoplasma infection remain insufficiently investigated. Therefore, in this study, we investigated the effect of infection with the flavescence doreé phytoplasma on the phenolic profile of grapevine (Vitis vinifera L.) leaves. Infection with the flavescence doreé phytoplasma was confirmed in symptomatic plants using the multiplex real-time PCR assay. The content of groups of phenolic metabolites was evaluated in grapevine leaves sampled at the three time points during the development of infection. Results revealed the lower content of most groups of phenolic compounds at the first time point. However, the content of total phenolic acids was higher at the first time point in leaves of infected plants compared to the uninfected leaves. At the third time point, content of all phenolics groups was higher in infected plants. Results point to induction of phenolic biosynthesis in infected plants at a late stage of infection, pointing to phenolic compounds as a biomarker of late stages of grapevine infection with the flavescence doreé phytoplasma.

Broccoli (Brassica oleracea botrytis var. cymosa) was grown to a stage with two adult leaves, after which it was subjected to temperature stress until the stage of development with 6-8 adult leaves. Ice water treatment was performed by applying ice to the substrate in pots in which broccoli was grown and hot water treatment by watering the substrate with water at a temperature of 80°C. A control group of plants was watered with room temperature water. After collecting the aboveground part of the plants, it was immediately frozen in a stream of liquid nitrogen and then lyophilized. The extracts were prepared in 70% ethanol, after which they were hydrolysed using 1.2 M hydrochloric acid. Separation, identification and quantification of vitamin C, ferulic and synapic acid and flavonols quercetin and kaempferol were performed using high performance liquid chromatography (HPLC). Stress caused by high temperature water significantly increased the concentration of vitamin C and ferulic acid and decreased the concentration of kaempferol and synapic acid in young broccoli plants. Stress caused by ice water did not increase the concentration of any of the analysed bioactive compounds, but therefore significantly reduced the concentration of kaempferol and synapic acid. The concentration of flavonol quercetin in young broccoli did not change significantly after stress caused by hot and ice water.

Plants challenged with high temperature (HT) undergo many adaptive mechanisms at molecular levels to keep normal physiological function. Such adaptations consequently influence their bioactivity. In this work, within the scope of the TEMPHYS project (IP-2020-02- 7585), we investigated the effect of HT on the antidiabetic activity of broccoli (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.) seedlings. The activity was spectrophotometrically assessed via the percentage of inhibition of α-amylase and α-glucosidase, enzymes required for carbohydrate digestion. Enzyme inhibitory activity was calculated from the equation: % inhibition = 100 – [(At – Atb / Ac – Acb) x 100], where At was absorbance of test, Atb was absorbance of test blank, Ac was absorbance of control, and Acb was absorbance of control blank. Seedlings were grown in a climatic chamber with temperature range 23°C/16h, 18°C/8h for control group, and 38°C/16h, 33°C/8h for HT group. The extracts were prepared from freeze- dried plant material using boiling (100°C) water followed by incubation on rotary extractor for 1 hour at 23°C. The results showed that broccoli seedlings aqueous extracts were more efficient in the inhibition of α-amylase (88% relative to the standard acarbose), than α-glucosidase (67% relative to the standard acarbose). HT significantly (p≤0.05) decreased the potential to inhibit α-amylase (80%), however it improved the potential to inhibit α-glucosidase (70%). These results show that high growing temperature significantly changes the biological effects of plants ; moreover, their activity toward different enzymes is specifically affected. Further more detailed analyses into the effect of global warming on cruciferous (Brassicaceae) vegetables bioactivity are needed.

Due to climate change, plants are exposed to abiotic stress, especially high temperatures. To combat those stressful conditions, they developed antioxidant mechanisms which help them to reduce reactive oxygen species. To investigate how high temperature affects the antioxidant potential of broccoli seedlings (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.), in the winter of 2021/2022, within the scope of the TEMPHYS project (IP-2020-02-7585), we cultivated three biological replicas of broccoli seedlings at high temperature (38 °C day/33 °C night) and three biological replicas at regular temperature (23 °C day/18 °C night) as a control group. Extracts of lyophilized broccoli tissue were made in 70% ethanol, and antioxidant potential was measured using four methods: ABTS, DPPH, FRAP and β- carotene bleaching. The obtained values were compared to those of standard Trolox solution of the same concentration as our extracts (i.e. 30 mg/mL for all the methods, except for FRAP which was 15 mg/mL). Based on the results of ABTS, FRAP and β-carotene bleaching method, high temperature significantly increased antioxidant potential of broccoli seedlings. However, DPPH method indicated a reduction of antioxidant potential. All the methods were significantly positively correlated with each other, except the DPPH which was negatively correlated with other methods. Both, at high and regular temperature, statistical methods principal component analysis and hierarchical clustering revealed higher similarity between second and third biological replicas, and separation of first biological replica. However, all biological replicas grown at high temperature tended to cluster together and clearly separated from those at regular temperature. In conclusion, based on three out of four used methods, high temperature significantly increased antioxidant potential of broccoli seedlings, suggesting further research into the effect on plant metabolism.

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 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.

Seedlings of cruciferous plants are becoming more represented in human diet and are often labelled as functional food because of their richness in healthy antioxidative compounds. In this study, we analysed the antioxidative potential of extracts from two types of cruciferous seedlings, broccoli (Brassica oleracea var. italica) and kale (B. oleracea var. acephala). Ethanolic extracts were subjected to in vitro simulated human digestion, and the antioxidative capacity of the extracts before and after salivary, gastric and intestinal phase of digestion was assessed using ABTS, DPPH and FRAP assays. ABTS assay revealed the highest antioxidative potential of both broccoli and kale seedlings extracts in the intestinal phase, pointing to the high gastrointestinal stability of antioxidative compounds from broccoli and kale extracts. Each of the three assays revealed the higher antioxidative potential of kale extract before and after in vitro digestion in comparison to the antioxidative potential of broccoli extract, pointing to kale seedlings being a richer source of compounds with high antioxidative activity. The results support the use of cruciferous seedlings as a natural source of compounds with high gastrointestinal stability and high antioxidative potential, pointing to their potential healthy bioactive properties in vivo.

Phytoplasmas, bacteria of genus ‘Candidatus Phytoplasma’, infect both plants and insects, which act as their vectors. They are pathogens of various agriculturally important plants, causing severe damage to crop. Phytoplasmas have been identified as causative agents of grapevine yellows (GY) diseases, which affect viticultural production worldwide. Two phytoplasmas have been linked to GY, one of which is the flavescence dorée phytoplasma (FDp, ribosomal group 16SrV), a quarantine pathogen in many countries. However, due to the inability to establish in vitro phytoplasma culture, phytoplasma research is still challenging. In this study, we collected symptomatic and asymptomatic leaves of grapevine (Vitis vinifera L.) var. ‘Pinot noir’ from a vineyard in central continental Croatia at three time points during the development of infection. Using a multiplex real time PCR assay, we confirmed the infection with FDp in symptomatic leaves. To assess the physiological response of grapevine to FDp infection, we used a plethora of spectrophotometric assays. Effect of FDp infection on photosynthetic pigment content in grapevine and level of proline and soluble sugars was analysed. Furthermore, the content of hydrogen peroxide, a reactive oxygen species (ROS), was assessed in sampled leaves, as well as the level of lipid peroxidation, an indicator of membrane damage. Results revealed an induction of ROS synthesis as well as the higher level of lipid peroxidation in leaves of infected plants, pointing to the occurrence of oxidative stress in infected plants. Induction of proline and sugar synthesis was also observed in infected plants, pointing to the activation of grapevine defence mechanisms against FDp infection. However, the biggest differences in the content between infected and uninfected leaves were observed for photosynthetic pigments, which point to a strong inhibition of photosynthetic processes in grapevine infected with FDp. Results expand the knowledge of the effect of phytoplasma infection on grapevine leaves physiology, as well as grapevine protective mechanisms against phytoplasma infection.

Phytoplasmas (genus Candidatus Phytoplasma) are a diverse group of bacterial intracellular pathogens causing significant damage in agriculture worldwide. However, due to inability of obtaining a pure phytoplasma culture in vitro, they remain insufficiently investigated. With the aim of studying the interactions between plants and phytoplasmas, grapevine (Vitis vinifera ; Vv) and flavescence dorée phytoplasma (FDp ; ribosomal group 16SrV), one of the main causative agents of grapevine yellows disease and a quarantine pathogen in Europe, were chosen as a model pathosystem. Leaves of symptomatic and asymptomatic plants from Continental Central Croatia were sampled three times during the development of symptoms. Using triplex real-time PCR assay, infection of Vv by FDp was confirmed in symptomatic plants. To assess the genetic structure of phytoplasmas, map gene was amplified and sequence d followed by phylogenetic analysis. In order to investigate changes in concentration of specialized metabolites between infected and uninfected Vv leaves, high performance liquid chromatography and spectrophotometric assays were used. Two different mapFD-genotypes (M38 and M54) were identified among analysed samples. Metabolomic analyses revealed a high dynamics of specialized metabolite concentrations (metabolite groups and individual compounds) during the development of infection. Furthermore, results point to a phytoplasma genotype-dependant effect on specialized metabolism of infected Vv leaves, as suggested by different patterns of change in phenolics concentration in investigated time points between plants infected with two different phytoplasma genotypes. The results contribute to the understanding of grapevine response to infection with different FDp genotypes and reveal potential protective mechanisms of Vv against phytoplasma infection.

Mature Brassicaceae plants are established as a great source of antioxidants, particularly phenolic compounds. Their seedlings, nowadays often branded as microgreens, also emerged as a rich source of healthy phytochemicals. However, results and comparisons of phenolic profiles of different Brassicaceae varieties are scarce. Therefore, in this study, ethanolic extracts of broccoli (Brassica oleracea var. italica), cauliflower (B. oleracea var. botrytis) and kale (B. oleracea var. acephala) were subjected to the chromatographic analysis using reversed-phase high-performance liquid chromatography (RP-HPLC). Using targeted metabolomics approach, we identified and quantified five phenolic acids (p- coumaric, ferulic, caffeic, sinapic and gallic acid) and two flavonoids (quercetin and kaempferol). In broccoli seedlings, highest levels of caffeic and gallic acid were recorded, as well as the highest quercetin content among the tested extracts. Cauliflower seedlings had the highest levels of kaempferol, ferulic and p-coumaric acid. Kale seedlings had the highest content of sinapic acid and quercetin. Results established that the phenolic profiles of different Brassica microgreens largely vary among cultivars. Each of the identified compounds were present in high concentrations in the extracts, confirming the cruciferous microgreens as a rich source of phenolic compounds. High content of flavonoids and phenolic acids contribute to the antioxidative potential of Brassica microgreens, establishing them as a healthy functional food which should be included in the human diet on a wider scale. Further studies should evaluate the health benefits of the Brassica microgreens in more detail.

The world community is making great efforts to understand the current and potential impacts of global warming on plants and animals. Not only because of the negative effects on crop production and therefore on food supply, but also because of changes in the nutritional value of vegetables that occur as a result of the temperature increase. Plants challenged with high temperature undergo many adaptive mechanisms at molecular levels to keep normal physiology function. They can adapt to different temperatures by changing their transcriptome, proteome and metabolome, or even by activating cell death mechanisms. Such adaptability is due to their intensive shifts in biochemical pathways, which, as a consequence, can significantly change their bioactivity as well. Changes like these could have significant outcome on the plant nutritional value and present serious challenges for global food security. Because of that the objective of our work is to investigate the consequences of environmental increased temperature on vegetable nutritional value. We are trying to assign correlations between temperature shift and plant biochemical traits employing targeted specific metabolomics approach, statistical data analyses and modelling in order to construct a model showing a tentative pattern of environment temperature effect on plant bioactive compounds. As a model plant organism we use broccoli (Brassica oleracea L. convar. botrytis (L.) Alef. Var. cymosa Duch.) in different stage of development.

The main goal of our study was to evaluate the effect of proteins (40 g/L casein), carbohydrates (25 g/L glucose) and oils (5% olive oil) on phytochemical bioavailability, antioxidant, antidiabetic and weight loss activity of gingko leaves standardized extracts after in vitro gastrointestinal digestion. Spectrophotometric methods were used to determine the concentration of total phytochemicals (TP: total phenols, TF: total flavonoids, TPA: total phenolic acids, TFLA: total flavanols, TFLO: total flavonols, TPAN: total proanthocyanidins and TTL: total triterpene lactones), antioxidant (DPPH and FRAP), antidiabetic (inhibition of α-glucosidase and BSA glycation) and weight loss (inhibition of lipase) activity. Ginkgo olive oil formulation had the highest TFLO, TFLA, TPAN and TTL bioavailability after in vitro gastrointestinal digestion. The highest TF and TPA bioavailability had gingko casein formulation. In the gingko water formulations, the highest TP bioavailability was detected. All gingko formulations had high (>70%) antioxidant activity (DPPH and FRAP), high (>70%) enzyme (α-glucosidase and lipase) inhibitory activity and high (>70%) inhibition of BSA glycation after in vitro digestion. According to our results, ginkgo formulation with olive oil has proved to be the formulation with the highest bioavailability of polyphenolic groups and terpene lactones after in vitro gastrointestinal digestion.

Cruciferous plants (family Brassicaceae) are known as a great source of healthy phytochemicals. In recent years, focus shifted from mature plants to seedlings of cruciferous plants. They emerged as a rich source of phenolic compounds and glucosinolates. However, detailed analysis of their bioactivity is still lacking. Considering a rise in prevalence of chronic illnesses due to modern lifestyle, plant foods are considered as food supplements because of the richness in healthy compounds. Of particular interest are phytochemicals with high antioxidative capacity. In this study, antioxidative potential of broccoli (Brassica oleracea var. italica), cauliflower (B. oleracea var. botrytis) and kale (B. oleracea var. acephala) seedlings extracts was assessed by ABTS, DPPH and FRAP assays. Results of the FRAP assay revealed that kale seedlings extract had the highest antioxidative capacity of all three extracts, with no difference between broccoli and cauliflower extracts. ABTS assay revealed that kale and cauliflower seedlings extract had higher antioxidative capacity than broccoli seedlings extract. DPPH assay did not show statistically significant difference in antioxidative capacity of three assayed extracts. Considering that two assays revealed that kale extract had the highest antioxidative capacity, this extract should be further evaluated for its bioactivity. Nevertheless, all three Brassica seedlings extracts showed high antioxidative capacity in vitro. Therefore, their intake as food supplements with high antioxidative potential is highly recommended. Further studies should aim to identify compounds from seedlings extract that contribute to antioxidative capacity of extracts the most, and evaluate their antioxidative properties in vivo.

Phytoplasmas, bacteria belonging to the genus ‘Candidatus Phytoplasma’, are known to be pathogenic for a wide variety of plant species. One of highly impacted plant cultures by phytoplasmas is grapevine (Vitis vinifera L.). Grapevine yellows are serious diseases affecting viticultural production worldwide. One of them is caused by the flavescence dorée phytoplasma (ribosomal group 16SrV), which is spread by the vector, leafhopper Scaphoideus titanus. Phytoplasmas are known to secrete effector molecules, which affect the physiology of their hosts, in order to increase their replication and spread. It is known that the flavescence dorée phytoplasma heavily affects photosynthetic pigments of grapevine, causing severe chlorosis in infected leaves. In this study, we sampled the leaves of symptomatic and asymptomatic plants to evaluate the dynamics of photosynthetic pigment content during the development of infection. Using real-time PCR, we confirmed the infection with the flavescence dorée phytoplasma in symptomatic plants and quantitatively determined the phytoplasma titre. Additionally, by amplification, sequencing and phylogenetic analysis of map gene amplicons, we detected two flavescence dorée genotypes in sampled grapevine leaves, M38 and M54, both belonging to the mapFD2 cluster. Spectrophotometrically, we analysed the effect of the flavescence dorée phytoplasma infection on photosynthetic pigment content in grapevine and its dynamics during the development of infection and whether it is impacted by phytoplasma genotype. Results revealed that M38 impacted photosynthetic pigments more severely than M54 genotype. Interestingly, M38 was present in lower count in grapevine leaves than the M54. However, for both genotypes, it was revealed that the highest reduction in photosynthetic pigments was at the second time point, which was also when the phytoplasma titre was the highest. This potentially links the phytoplasma titre with severity of chlorosis symptoms due to the degradation of photosynthetic pigments. These results contribute to resolving the mechanisms of flavescence dorée phytoplasma pathogenicity on the level of photosynthetic pigments and provide details for the search of potential flavescence dorée phytoplasma effectors which target photosynthetic pigments.

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.

Climate change is a global problem that has changed both the physiological and biochemical parameters of plants. Considering that plants are sessile organisms, their survival depends on the effective activation of metabolic mechanisms of resistance to such stress. As part of this study, we investigated the impact of low and high temperature water on the content of chlorophyll, lycopene, carotenoids and porphyrins in broccoli (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.). For all pigments, except chlorophyll a, a statistically significant difference was observed between the group treated with water of high temperature and the remaining two groups (control and group treated with water of low temperature). In the group treated with water of high temperature, the highest proportion of chlorophyll b (5.35 ± 0.34 mg/g of dry weight (sm)), lycopene (0.28 ± 0.016 mg/g sm), and porphyrins (28.27 ± 1.09 mg/g sm) was recorded. The proportion of carotenoids, on the other hand, was the lowest in this group (0.40 ± 0.19 mg/g sm). Based on this, we conclude that chlorophyll b, lycopene, porphyrins and carotenoids of broccoli are significantly more sensitive to high temperature water stress than to low temperature water.

Due to climate change, sudden and intense changes in weather conditions are becoming more frequent. Sessile organisms such as plants cannot "escape" from high or low temperatures but adapt their metabolism to new conditions to survive. Oxidative stress is a common response of plants exposed to extreme temperatures. During temperature stress, the production of a large amount of ROS can be a major risk factor for plant cells and therefore stimulates the synthesis of antioxidants in them. As part of this paper, we investigated the impact of cold (ice) and hot water (80°C) on the antioxidant potential of broccoli (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.) in the developmental stage with 6-8 leaves. We used three methods of analysis, ABTS, FRAP and DPPH. The results showed that none of the treatments significantly changed the antioxidant potential measured by ABTS and FRAP method, however, both caused a decrease in potential as measured by the DPPH method. In particular, the control group accounted for 4.71 ± 0.40 % of radical inhibition compared to Trolox, the group treated with cold water for 4.13 ± 0.51 %, and the group treated with hot water for 3.69 ± 0.46 %. Such a difference in results between different methods is a consequence of different types of antioxidants present in samples that react differently with the radicals used. Based on these results, we conclude that using cold or hot water, we cannot improve the antioxidant potential of broccoli ; moreover, according to the DPPH method, this potential was reduced.

The goal of our study was to evaluate the effect of high (HT: 38°C day/33°C night) and regular (RT: 23°C day/18°C night) growing temperature on the bioavailability of total glucosinolates and phenolics from broccoli (Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch) sprouts upon final, intestinal, phase of in vitro simulated human digestion. The extracts were prepared from freeze-dried plant material using boiling (100°C) water followed by incubation on rotary extractor for 1 hour at 23°C. The results showed that HT negatively affected the intestinal bioavailability of total glucosinolates from broccoli sprouts, i.e. their concentration was decreased to 47 % of the initial concentration (non-digested extract). On the other hand, RT did not change the bioavailability of total glucosinolates significantly (p≤0.05). Total phenolics bioavailability was not affected neither by HT, nor by RT condition ; in both cases it was very high (>90 %). These results show that glucosinolates of broccoli sprouts are significantly more susceptible to HT than total phenolics, and suggests future more detailed analyses of Brassicaceae microgreens phytochemical temperature susceptibility/resistance.

Vegetables from cruciferous plants (family Brassicaceae) are known as a great source of healthpromoting chemicals. In recent years, focus shifted from mature plants to seedlings of cruciferous plants, which are nowadays often branded as microgreens. They emerged as a rich source of phenolics and glucosinolates. However, detailed analysis of their bioactivity is still lacking. Considering a rise in prevalence of chronic illnesses, plant foods are considered as food supplements that could be used in fighting illnesses such as type II diabetes and obesity. Gastrointestinal enzymes, such as α-amylase, α- glucosidase and pancreatic lipase are often targeted in treatment of those diseases. Search for inhibitors of these enzymes continues, with plant foods being a potential source. In this study, antidiabetic and anti-obesity potential of broccoli (Brassica oleracea var. italica), cauliflower (B. oleracea var. botrytis) and kale (B. oleracea var. acephala) seedlings was assessed by testing the inhibitory capacity of their ethanolic extract on α-amylase, α-glucosidase and pancreatic lipase in vitro. Results revealed that broccoli seedlings had the highest inhibitory capacity against α-amylase, and kale seedlings had the highest inhibitory capacity against α- glucosidase. Kale and cauliflower had a similar inhibitory capacity against pancreatic lipase, while inhibitory capacity of broccoli extracts was lower. However, neither had an inhibitory capacity higher than standard compounds used as inhibitors of these enzymes. Nevertheless, extracts of broccoli, cauliflower and kale seedlings extracts could be used as supplements in treating diseases like diabetes and obesity, considering they possess inhibitory capacity against α-amylase, α- glucosidase and pancreatic lipase. Further studies should aim to identify compounds from seedlings extract that contribute to inhibitory capacity of those enzymes the most, and evaluate their antidiabetic and anti-obesity properties in vivo.

Phytoplasmas (genus ‘Candidatus Phytoplasma’) are uncultivable bacterial intracellular parasites of plants, as well as insects, which act as vectors for their dispersal. They cause heavy damage to agricultural production worldwide. Among infecting other agriculturally important plant species, they are known to heavily affect viticultural production. Two phytoplasmas have been identified as causative agents of grapevine yellows diseases, one of which is the flavescence dorée phytoplasma (FDp), a quarantine pathogen in many countries. Due to the inability to cultivate phytoplasmas in a pure culture in vitro, phytoplasma research faces many challenges. In this study, we sampled the leaves from symptomatic and asymptomatic grapevine (Vitis vinifera L.) var. ‘Pinot noir’ from a vineyard in central continental Croatia at three time points during the development of infection. Infection of symptomatic plants with FDp was successfully confirmed using a multiplex real-time PCR assay, developed for simultaneous detection of both phytoplasma species causing the grapevine yellows disease. In order to analyse and compare the phenolic content of infected and uninfected grapevine leaves, extraction of phenolic compounds was carried out with 70% (v/v) ethanol. Using various spectrophotometric assays, content of total phenolics, flavonoids, hydroxycinnamic acids, flavanols, catechins, proanthocyanidins and tannins was analysed. Results revealed a high dynamic of the content of analysed groups of phenolic compounds in grapevine infected by the FDp through the development of infection. Several groups of phenolic compounds whose content increased in infected leaves compared to uninfected were identified as potential protective compounds against FDp infection. Severe changes to the phenolic compounds content in infected grapevine leaves compared to the uninfected leaves point the phenolics biosynthesis being a target of FDp effector molecules which interfere with the host physiological processes, because of the potential of phenolic compounds as protective molecules against phytoplasma infection.

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 principal component analysis (PCA) was used to gain insight into the relationship between phytochemicals (total phenols - TP, total phenolic acid - TPA, total flavonoids -TF, total anthocyanidins - TA ; total hydroxycinnamic acids - THA, and total flavanols - TFL, glucosinolates – Gls, sugar – TS) and antioxidant, antidiabetic and anti-obesity activity in three developmental stages of broccoli after in vitro digestion. Different developmental stages of broccoli (1st stage: seedlings, 2nd stage: plants with 2 real leaves, and 3rd stage: plants with 4-6 real leaves) were collected in July and August 2021, and extracts were prepared in hot 70% ethanol. In PCA the first and the second principal components described 64.62% and 35.38% of the variance after in vitro digestion, respectively. Together, the first two PCs represent 100% of the total variability. After digestion, 1st developmental stage of broccoli had strong loadings with most of the tested chemicals (TA, Gls, TS), DPPH and inhibition of alpha-amylase. Broccoli 2nd developmental stage had strong loadings with TPA and inhibition of lipase, while 3rd developmental stage had strong loadings with TF and inhibition of alpha-glucosidase. Pearson’s correlation coefficient between phytochemicals, antioxidant (ABTS, DPPH and FRAP), antidiabetic (inhibition of alpha-amylase and alpha-glucosidase) and anti-obesity activity (lipase inhibition) was also performed. After digestion, TP correlated very strongly (0.999) with THA, TPA correlated very strongly with antioxidant (FRAP: 0.998) and anti-obesity activity (inhibition of lipase: 1.000), TA correlated very strongly alpha1.000) with antidiabetic activity (inhibition of alpha-amylase), Gls correlated very strongly with antioxidant activity (DPPH: 0.999), and DPPH correlated very strongly (0.997) with alpha-amylase. According to the results after in vitro digestion, 1st developmental stage of broccoli was the best source of TA, Gls and TS, that are closely related with antioxidant activity (DPPH) and key enzyme linked to type-2 diabetes (alpha-amylase). 2nd developmental stage of broccoli was the best source of TPA, that are closely related with inhibition of key enzyme linked to obesity (lipase) ; and 3rd developmental stage was the best source of TF, that are closely related with inhibition of key enzyme linked to type-2 diabetes (alpha-glucosidase).

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.

Invasive alien plant species are a rich reservoir of specialised metabolites, mainly phenolic compounds. Phenolics have a common origin in the phenylpropanoid biosynthetic pathway and are responsible for numerous protective and antioxidant properties. Here we present the preliminary research results of the Croatian Science Foundation project NATURALLY (IP-2020-02-6899), intending to explore the potential of natural extracts of alien invasive plant species for potential application as phytopharmaceuticals. In our study, we used the method of high-performance liquid chromatography with reversed phases (RP-HPLC) to perform tentative profiling and chemical characterisation of 70 % ethanol leaf extracts of four invasive species: Ailanthus altissima (Mill.) Swingle, Helianthus tuberosus L., Robinia pseudoacacia L. and Solidago canadensis L., all included in the list of 70 most invasive plant species in Croatia. Preliminary results of RP-HPLC extracts profiling indicate the phytochemical diversity of analysed species. The most common detected compounds were vitamin C (L-ascorbic acid), phenolic acids (gallic, benzoic, chlorogenic, vanillic, caffeic, p-coumaric, synapic, and ferulic acid), and flavonoids (quercetin, kaempferol and isorhamnetin). Vitamin C was detected in all analysed species, with the highest concentration in R. pseudoacacia (2789, 00 mg/kg DW) and the lowest in H. tuberosus (799.96 mg/kg DW). One of the most represented phenolic acids was chlorogenic acid, most abundant in H. tuberosus (11906, 93 mg/kg DW). Synapic acid, however, was detected only in R. pseudoacacia (11, 23 mg/kg DW). Kaempferol stands out with high concentrations among the detected flavonoids, especially in S. canadensis (17173, 56 mg/kg DW). Future research steps will involve extracts fractionation, as well as the comparison of the antioxidant properties of whole extracts and isolated fractions. Obtained results indicate the abundance of bioactive compounds in investigated invasive species and suggest their potential as a source of natural antioxidants in the food industry.