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The present study aimed to develop a system using a combination of enzymatic and microbial degradation techniques for removing phenol from contaminated water. In our prior research, the HRP enzyme extracted from horseradish roots was utilized within a core-shell microcapsule to reduce phenolic shock, serving as a monolayer column. To complete the phenol removal process, a second column containing degrading microorganisms was added to the last column in this research. Phenol-degrading bacteria were isolated from different microbial sources on a phenolic base medium. Additionally, encapsulated calcium peroxide nanoparticles were used to provide dissolved oxygen for the microbial population. Results showed that the both isolated strains, WC1 and CC1, were able to completely remove phenol from the contaminated influent water the range within 5 to 7 days, respectively. Molecular identification showed 99.8% similarity for WC1 isolate to Stenotrophomonas rizophila strain e-p10 and 99.9% similarity for CC1 isolate to Bacillus cereus strain IAM 12,605. The results also indicated that columns using activated sludge as a microbial source had the highest removal rate, with the microbial biofilm completely removing 100% of the 100 mg/L phenol concentration in contaminated influent water after 40 days. Finally, the concurrent use of core-shell microcapsules containing enzymes and capsules containing Stenotrophomonas sp. WC1 strain in two continuous column reactors was able to completely remove phenol from polluted water with a concentration of 500 mg/L for a period of 20 days. The results suggest that a combination of enzymatic and microbial degrading systems can be used as a new system to remove phenol from polluted streams with higher concentrations of phenol by eliminating the shock of phenol on the microbial population., (© 2024. The Author(s).)

β-Glucosidases play an important role in the chemical defense of many insects by hydrolyzing and thereby activating glucosylated pro-toxins that are either synthesized de novo or sequestered from the insect's diet. The horseradish flea beetle, Phyllotreta armoraciae, sequesters pro-toxic glucosinolates from its brassicaceous host plants and possesses endogenous β-thioglucosidase enzymes, known as myrosinases, for glucosinolate activation. Here, we identify three myrosinase genes in P. armoraciae (PaMyr) with distinct expression patterns during beetle ontogeny. By using RNA interference, we demonstrate that PaMyr1 is responsible for myrosinase activity in adults, whereas PaMyr2 is responsible for myrosinase activity in larvae. Compared to PaMyr1 and PaMyr2, PaMyr3 was only weakly expressed in our laboratory population, but may contribute to myrosinase activity in larvae. Silencing of PaMyr2 resulted in lower larval survival in a predation experiment and also reduced the breakdown of sequestered glucosinolates in uninjured larvae. This suggests that PaMyr2 is involved in both activated defense and the endogenous turnover of sequestered glucosinolates in P. armoraciae larvae. In activity assays with recombinant enzymes, PaMyr1 and PaMyr2 preferred different glucosinolates as substrates, which was consistent with the enzyme activities in crude protein extracts from adults and larvae, respectively. These differences were unexpected because larvae and adults sequester the same glucosinolates. Possible reasons for different myrosinase activities in Phyllotreta larvae and adults are discussed., Competing Interests: Declaration of competing interest None., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Armoracia rusticana P. G. Gaertner. belongs to the Brassicaceae family and has aroused scientific interest for its anti-inflammatory and anticancer activities. In a continuing investigation to discover bioactive constituents from A. rusticana, we isolated 19 phenolic glycosides including three undescribed flavonol glycosides and one undescribed neolignan glycoside from MeOH extract of this plant. Their structures were elucidated based on NMR spectroscopic analysis ( 1 H, 13 C, 1 H- 1 H COSY, HSQC, and HMBC), HRESIMS, and chemical methods. The determination of their absolute configuration was accomplished by ECD and LC-MS analysis. All the compounds were assessed for their potential neurotrophic activity through induction of nerve growth factor in C6 glioma cell lines and for their anti-neuroinflammatory activity based on the measurement of inhibition levels of nitric oxide production and pro-inflammatory cytokines (i.e., IL-1β, IL-6, and TNF-α) in lipopolysaccharide-activated microglia BV-2 cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Main Conclusion: We characterized an efficient chimeric sub-genomic transcript promoter from Horseradish Latent Virus, FHS4, active in both dicot and monocot plants, and it could be a potential tool for plant biotechnology. Plant pararetroviruses are a rich source of novel plant promoters widely used for biotechnological applications. Here, we comprehensively characterized a unique sub-genomic transcript (Sgt) promoter of Horseradish Latent Virus (HRLV) and identified a fragment (HS4; - 340 to + 10; 351 bp) that showed the highest expression of reporter genes in both transient and transgenic assays as evidenced by biochemical, histochemical GUS reporter assay and transcript analysis of uidA gene by qRT-PCR. Phylogenetic analysis showed that the HSgt promoter was closely related to the sub-genomic promoter of the Cauliflower Mosaic Virus (CaMV19S). We found that the as-1 element and W-box played an important role in the transcriptional activity of the HS4 promoter. Furthermore, the HS4 promoter was also induced by salicylic acid. Alongside, we enhanced the activity of the HS4 promoter by coupling the enhancer region from Figwort Mosaic Virus (FMV) promoter to the upstream region of it. This hybrid promoter FHS4 was around 1.1 times stronger than the most commonly used promoter, 35S (Cauliflower Mosaic Virus full-length transcript promoter), and was efficient in driving reporter genes in both dicot and monocot plants. Subsequently, transgenic tobacco plants expressing an anti-microbial peptide BrLTP2.1 (Brassica rapa lipid transport protein 2.1), under the control of the FHS4 promoter, were developed. The in vitro anti-fungal assay revealed that the plant-derived BrLTP2.1 protein driven by an FHS4 promoter manifested increased resistance against an important plant fungal pathogen, Alternaria alternata. Finally, we concluded that the FHS4 promoter can be used as an alternative to the 35S promoter and has a high potential to become an efficient tool in plant biotechnology., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Horseradish degradation products, mainly isothiocyanates (ITC) and nitriles, along with their precursors glucosinolates, were characterized by GC-MS and UHPLC-MS/MS, respectively. Volatiles from horseradish leaves and roots were isolated using microwave assisted-distillation (MAD), microwave hydrodiffusion and gravity (MHG) and hydrodistillation (HD). Allyl ITC was predominant in the leaves regardless of the isolation method while MAD, MHG, and HD of the roots resulted in different yields of allyl ITC, 2-phenylethyl ITC, and their nitriles. The antimicrobial potential of roots volatiles and their main compounds was assessed against sixteen emerging food spoilage and opportunistic pathogens. The MHG isolate was the most active, inhibiting bacteria at minimal inhibitory concentrations (MICs) from only 3.75 to 30 µg/mL, and fungi at MIC 50 between <0.12 and 0.47 µg/mL. Cytotoxic activity of volatile isolates and their main compounds were tested against two human cancer cell lines using MTT assay after 72 h. The roots volatiles showed best cytotoxic activity (HD; IC 50 = 2.62 μg/mL) against human lung A549 and human bladder T24 cancer cell lines (HD; IC 50 = 0.57 μg/mL). Generally, 2-phenylethyl ITC, which was tested for its antimicrobial and cytotoxic activities along with two other major components allyl ITC and 3-phenylpropanenitrile, showed the best biological activities.

Electron and proton transfer reactions in enzymes are enigmatic and have attracted a great deal of theoretical, experimental, and practical attention. The oxidoreductases provide model systems for testing theoretical predictions, applying experimental techniques to gain insight into catalytic mechanisms, and creating industrially important bio(electro)conversion processes. Most previous and ongoing research on enzymatic electron transfer has exploited a theoretically and practically sound but limited approach that uses a series of structurally similar ("homologous") substrates, measures reaction rate constants and Gibbs free energies of reactions, and analyses trends predicted by electron transfer theory. This approach, proposed half a century ago, is based on a hitherto unproved hypothesis that pre-exponential factors of rate constants are similar for homologous substrates. Here, we propose a novel approach to investigating electron and proton transfer catalysed by oxidoreductases. We demonstrate the validity of this new approach for elucidating the kinetics of oxidation of "non-homologous" substrates catalysed by compound II of Coprinopsis cinerea and Armoracia rusticana peroxidases. This study - using the Marcus theory - demonstrates that reactions are not only limited by electron transfer, but a proton is transferred after the electron transfer event and thus both events control the reaction rate of peroxidase-catalysed oxidation of substrates.

Horseradish hairy root cultures are suitable plant tissue organs to study the glucosinolate-myrosinase-isothiocyanate system and also to produce the biologically active isothiocyanates and horseradish peroxidase, widely used in molecular biology. Fifty hairy root clones were isolated after Agrobacterium rhizogenes infection of surface sterilized Armoracia rusticana petioles and leaf blades, from which 21 were viable after antibiotic treatment. Biomass properties (e.g. dry weight %, daily growth index), glucosinolate content (analyzed by liquid chromatography-electronspray ionization-mass spectrometry (LC-ESI-MS/MS)), isothiocyanate and nitrile content (analyzed by gas chromatography-mass spectrometry (GC-MS)), myrosinase (on-gel detection) and horseradish peroxidase enzyme patterns (on-gel detection and spectrophotometry), and morphological features were examined with multi-variable statistical analysis. In addition to the several positive and negative correlations, the most outstanding phenomenon was many parameters of the hairy root clones showed dependence on the organ of origin. Among others, the daily growth index, sinigrin, glucobrassicin, 3-phenylpropionitrile, indole-3-acetonitrile and horseradish peroxidase values showed significantly higher levels in horseradish hairy root cultures initiated from leaf blades., Competing Interests: The authors declare no conflict of interest.

The potential contamination of the food chain is the most important aspect of arsenic (As) pollution, since it is highly toxic to all organisms. Thus, the search for As hyperaccumulators suitable to remove As from contaminated soils appears to be a vital task. Horseradish (Armoracia rusticana), a crop plant with a high potential to accumulate heavy metals, can also serve to study the physiological processes that accompany arsenic stress. The significant adverse effect caused by arsenic exposure is an oxidative stress. Plants have developed a highly organized system to quench free radicals, which includes the action of both enzymatic and non-enzymatic quenching. Saccharides are proposed to possess outstanding antioxidant activity in vitro, and thus, they are likely to effectively quench free radicals also in plant tissues. In this study, root cultures (hairy root type) of horseradish were grown in vitro on media with different concentrations of arsenic (5-60 µg l -1 ). Arsenic slowed down the growth, nevertheless up to three-fold biomass increase was achieved at the highest dose. Moreover, root tissues were able to remove as much as 75% of arsenic from the cultivation medium within 7 days. We also evaluated diverse oxidative-stress-related features: contents of reactive oxygen species, the activities of key antioxidant enzymes, and the contents of important antioxidant molecules, such as glutathione, proline, phenolic compounds and non-structural carbohydrates. At all arsenic treatments, we observed a significant proline content increase and enhanced antioxidant enzymes (peroxidase, catalase and glutathione-S-transpherase) activities peaking, however, at different doses. Soluble carbohydrates contents also significantly increased after 7-day treatment a then decreased nearly to the original levels. This study points to efficient antioxidant system of horseradish hairy roots enabling good growth and substantial As accumulation even under high As exposure. Providing that horseradish shares these important features with this model system, we could propose that horseradish is a promising candidate to exploit in arsenic phytoremediation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The usage of insects as model organisms is becoming more and more common in toxicological, pharmacological, genetic and biomedical research. Insects, such as fruit flies ( Drosophila melanogaster ), locusts ( Locusta migratoria ), stick insects ( Baculum extradentatum ) or beetles ( Tenebrio molitor ) are used to assess the effect of different active compounds, as well as to analyse the background and course of certain diseases, including heart disorders. The goal of this study was to assess the influence of secondary metabolites extracted from Solanaceae and Brassicaceae plants: Potato ( Solanum tuberosum ), tomato ( Solanum lycopersicum ), black nightshade ( Solanum nigrum ) and horseradish ( Armoracia rusticana ), on T. molitor beetle heart contractility in comparison with pure alkaloids. During the in vivo bioassays, the plants glycoalkaloid extracts and pure substances were injected at the concentration 10 -5 M into T. molitor pupa and evoked changes in heart activity. Pure glycoalkaloids caused mainly positive chronotropic effects, dependant on heart activity phase during a 24-h period of recording. Moreover, the substances affected the duration of the heart activity phases. Similarly, to the pure glycoalkaloids, the tested extracts also mainly accelerated the heart rhythm, however S. tuberosum and S. lycopersicum extracts slightly decreased the heart contractions frequency in the last 6 h of the recording. Cardioacceleratory activity of only S. lycopersicum extract was higher than single alkaloids whereas S. tubersoum and S. nigrum extracts were less active when compared to pure alkaloids. The most cardioactive substance was chaconine which strongly stimulated heart action during the whole recording after injection. A. rusticana extract which is composed mainly of glucosinolates did not significantly affect the heart contractions. Obtained results showed that glycoalkaloids were much more active than glucosinolates. However, the extracts depending on the plant species might be more or less active than pure substances.

Background: The health of plants is heavily influenced by the intensively researched plant microbiome. The microbiome has to cope with the plant's defensive secondary metabolites to survive and develop, but studies that describe this interaction are rare. In the current study, we describe interactions of endophytic fungi with a widely researched chemical defense system, the glucosinolate - myrosinase - isothiocyanate system. The antifungal isothiocyanates are also of special interest because of their beneficial effects on human consumers., Results: Seven endophytic fungi were isolated from horseradish roots (Armoracia rusticana), from the genera Fusarium, Macrophomina, Setophoma, Paraphoma and Oidiodendron. LC-ESI-MS analysis of the horseradish extract incubated with these fungi showed that six of seven strains could decompose different classes of glucosinolates. Aliphatic, aromatic, thiomethylalkyl and indolic glucosinolates were decomposed by different strains at different rates. SPME-GC-MS measurements showed that two strains released significant amounts of allyl isothiocyanate into the surrounding air, but allyl nitrile was not detected. The LC-ESI-MS analysis of many strains' media showed the presence of allyl isothiocyanate - glutathione conjugate during the decomposition of sinigrin. Four endophytic strains also accepted sinigrin as the sole carbon source. Isothiocyanates inhibited the growth of fungi at various concentrations, phenylethyl isothiocyanate was more potent than allyl isothiocyanate (mean IC 50 was 2.30-fold lower). As a control group, ten soil fungi from the same soil were used. They decomposed glucosinolates with lower overall efficiency: six of ten strains had insignificant or weak activities and only three could use sinigrin as a carbon source. The soil fungi also showed lower AITC tolerance in the growth inhibition assay: the median IC 50 values were 0.1925 mM for endophytes and 0.0899 mM for soil fungi., Conclusions: The host's glucosinolates can be used by the tested endophytic fungi as nutrients or to gain competitive advantage over less tolerant species. These activities were much less apparent among the soil fungi. This suggests that the endophytes show adaptation to the host plant's secondary metabolites and that host metabolite specific activities are enriched in the root microbiome. The results present background mechanisms enabling an understanding of how plants shape their microbiome.

Carbon nanomaterials have emerged as suitable supports for enzyme immobilization and stabilization due to their inherently large surface area, high electrical conductivity, chemical stability, and mechanical strength. In this paper, carbon nano-onions (CNOs) were used as supports to immobilize alkaline phosphatase, horseradish peroxidase, and glucose oxidase. CNOs were first functionalized by oxidation to generate carboxylic groups on the surface followed by the covalent linking of using a soluble carbodiimide as coupling agent. The CNO-enzyme conjugates were characterized by transmission electron microscopy and Raman spectroscopy. Thermogravimetric analysis revealed a specific enzyme load of ∼0.5 mg of protein per milligram of CNO. The immobilized enzymes showed enhanced storage stability without altering the optimum pH and temperatures. These properties make the prepared nanobiocatalyst of potential interest in biosensing and other biotechnological applications.

Carbamazepine (CBZ) is a pharmaceutical frequently categorized as a recalcitrant pollutant in the aquatic environment. Endophytic bacteria previously isolated from reed plants have shown the ability to promote growth of their host and to contribute to CBZ metabolism. In this work, a horseradish (Armoracia rusticana) hairy root (HR) culture has been used as a plant model to study the interactions between roots and endophytic bacteria in response to CBZ exposure. HRs could remove up to 5% of the initial CBZ concentration when they were grown in spiked Murashige and Skoog (MS) medium. Higher removal rates were observed when HRs were inoculated with the endophytic bacteria Rhizobium radiobacter (21%) and Diaphorobacter nitroreducens (10%). Transformation products resulting from CBZ degradation were identified using liquid chromatography-ultra high-resolution quadrupole time of flight mass spectrometry (LC-UHR-QTOF-MS). CBZ metabolism could be divided in four pathways. Metabolites involving GSH conjugation and 2,3-dihydroxylation, as well as acridine related compounds are described in plants for the first time. This study presents strong evidence that xenobiotic metabolism and degradation pathways in plants can be modulated by the interaction with their endophytic community. Hence it points to plausible applications for the elimination of recalcitrant compounds such as CBZ from wastewater in CWs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Key Message: The promoter fragment described in this study can be employed for strong transgene expression under both biotic and abiotic stress conditions. Plant-infecting Caulimoviruses have evolved multiple regulatory mechanisms to address various environmental stimuli during the course of evolution. One such mechanism involves the retention of discrete stress responsive cis-elements which are required for their survival and host-specificity. Here we describe the characterization of a novel Caulimoviral promoter isolated from Horseradish Latent Virus (HRLV) and its regulation by multiple stress responsive Transcription factors (TFs) namely DREB1, AREB1 and TGA1a. The activity of full length transcript (Flt-) promoter from HRLV (- 677 to + 283) was investigated in both transient and transgenic assays where we identified H12 (- 427 to + 73) as the highest expressing fragment having ~ 2.5-fold stronger activity than the CaMV35S promoter. The H12 promoter was highly active and near-constitutive in the vegetative and reproductive parts of both Tobacco and Arabidopsis transgenic plants. Interestingly, H12 contains a distinct cluster of cis-elements like dehydration-responsive element (DRE-core; GCCGAC), an ABA-responsive element (ABRE; ACGTGTC) and as-1 element (TGACG) which are known to be induced by cold, drought and pathogen/SA respectively. The specific binding of DREB1, AREB1 and TGA1a to DRE, ABRE and as-1 elements respectively were confirmed by the gel-binding assays using H12 promoter-specific probes. Detailed mutational analysis of the H12 promoter suggested that the presence of DRE-core and as-1 element was indispensable for its activity which was further confirmed by the transactivation assays. Our studies imply that H12 could be a valuable genetic tool for regulated transgene expression under diverse environmental conditions.

Horseradish essential oil (HREO; a natural mixture of different isothiocyanates) had strong fungicide effect against Candida albicans both in volatile and liquid phase. In liquid phase this antifungal effect was more significant than those of its main components allyl, and 2-phenylethyl isothiocyanate. HREO, at sublethal concentration, induced oxidative stress which was characterized with elevated superoxide content and up-regulated specific glutathione reductase, glutathione peroxidase, catalase and superoxide dismutase activities. Induction of specific glutathione S-transferase activities as marker of glutathione (GSH) dependent detoxification was also observed. At higher concentration, HREO depleted the GSH pool, increased heavily the superoxide production and killed the cells rapidly. HREO and the GSH pool depleting agent, 1-chlore-2,4-dinitrobenzene showed strong synergism when they were applied together to kill C. albicans cells. Based on all these, we assume that GSH metabolism protects fungi against isothiocyanates., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The wide application of rare earth elements (REEs) have led to their diffusion and accumulation in the environment. The activation of endocytosis is the primary response of plant cells to REEs. Calmodulin (CaM), as an important substance in calcium (Ca) signaling systems, regulating almost all of the physiological activities in plants, such as cellular metabolism, cell growth and division. However, the response of CaM to endocytosis activated by REEs remains unknown. By using immunofluorescence labeling and a confocal laser scanning microscope, we found that trivalent lanthanum [La(III)], an REE ion, affected the expression of CaM in endocytosis. Using circular dichroism, X-ray photoelectron spectroscopy and computer simulations, we demonstrated that a low concentration of La(III) could interact with extracellular CaM by electrostatic attraction and was then bound to two Ca-binding sites of CaM, making the molecular structure more compact and orderly, whereas a high concentration of La(III) could be coordinated with cytoplasmic CaM or bound to other Ca-binding sites, making the molecular structure more loose and disorderly. Our results provide a reference for revealing the action mechanisms of REEs in plant cells., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Oxybenzone (OBZ), known as Benzophenone-3, is a commonly used UV filter in sun tans and skin protectants, entering aquatic systems either directly during recreational activities or indirectly through wastewater treatment plants discharge. To study the potential degradation capacity of plants for OBZ in phytotreatment, a well-established hairy root culture (Armoracia rusticana) was treated with OBZ. More than 20% of spiked OBZ (100μM) was eliminated from the medium by hairy roots after 3h of exposure. Two metabolites were identified as oxybenzone-glucoside (OBZ-Glu) and oxybenzone-(6-O-malonyl)-glucoside (OBZ-Mal-Glu) by LC-MS/MS and TOF-MS. Formation of these metabolites was confirmed by enzymatic synthesis, as well as enzymatic and alkaline hydrolysis. Incubation with O-glucosyltransferase (O-GT) extracted from roots formed OBZ-Glu; whereas β-d-Glucosidase hydrolyzed OBZ-Glu. However, alkaline hydrolysis led to cleavage of OBZ-Mal-Glu and yielded OBZ-Glu. In the hairy root culture, an excretion of OBZ-Glu into the growth medium was observed while the corresponding OBZ-Mal-Glu remained stored in root cells over the incubation time. We propose that metabolism of oxybenzone in plants involves initial conjugation with glucose to form OBZ-Glu followed by malonylation to yield OBZ-Mal-Glu. To our best knowledge this first finding presenting the potential of plants to degrade benzophenone type UV filters by phytoremediation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Rare earth elements, especially terbium (Tb), are high-valence heavy metal elements that accumulate in the environment, and they show toxic effects on plants. Signaling molecules regulate many physiological and biochemical processes in plants. How rare earth elements affect signaling molecules remains largely unknown. In the present study, the effects of Tb(3+) on some extracellular and intracellular signaling molecules (gibberellic acid, abscisic acid, auxin, H2O2, and Ca(2+)) in horseradish leaves were investigated by using high-performance liquid chromatography, X-ray energy spectrometry, and transmission electron microscopy, and Tb(3+) was sprayed on the surface of leaves. Tb(3+) treatment decreased the auxin and gibberellic acid contents and increased the abscisic acid content. These changes in the contents of phytohormones (gibberellic acid, abscisic acid, and auxin) triggered excessive production of intracellular H2O2. Consequently, the increase in H2O2 content stimulated the influx of extracellular Ca(2+) and the release of Ca(2+) from Ca(2+) stores, leading to Ca(2+) overload and the resulting inhibition of physiological and biochemical processes. The effects outlined above were more evident with increasing the concentration of Tb(3+) sprayed on horseradish leaves. Our data provide a possible underlying mechanism of Tb(3+) action on plants.

The pollution of the environment by rare earth elements (REEs) causes deleterious effects on plants. Peroxidase plays important roles in plant response to various environmental stresses. Here, to further understand the overall roles of peroxidase in response to REE stress, the effects of the REE terbium ion (Tb(3+)) on the peroxidase activity and H2O2 and lignin contents in the leaves and roots of horseradish during different growth stages were simultaneously investigated. The results showed that after 24 and 48 h of Tb(3+) treatment, the peroxidase activity in horseradish leaves decreased, while the H2O2 and lignin contents increased. After a long-term (8 and 16 days) treatment with Tb(3+), these effects were also observed in the roots. The analysis of the changes in peroxidase activity and H2O2 and lignin contents revealed that peroxidase plays important roles in not only reactive oxygen species scavenging but also cell wall lignification in horseradish under Tb(3+) stress. These roles were closely related to the dose of Tb(3+), duration of stress, and growth stages of horseradish.

It has long been observed that rare earth elements (REEs) regulate multiple facets of plant growth and development. However, the underlying mechanisms remain largely unclear. Here, using electron microscopic autoradiography, we show the life cycle of a light REE (lanthanum) and a heavy REE (terbium) in horseradish leaf cells. Our data indicate that REEs were first anchored on the plasma membrane in the form of nanoscale particles, and then entered the cells by endocytosis. Consistently, REEs activated endocytosis in plant cells, which may be the cellular basis of REE actions in plants. Moreover, we discovered that a portion of REEs was successively released into the cytoplasm, self-assembled to form nanoscale clusters, and finally deposited in horseradish leaf cells. Taken together, our data reveal the life cycle of REEs and their cellular behaviors in plant cells, which shed light on the cellular mechanisms of REE actions in living organisms.

The pungent taste of horseradish is caused by isothiocyanates which are released from glucosinolates by myrosinases. These enzymes are encoded by genes belonging to one of two subfamilies, termed MYR I and MYR II, respectively. A MYR II-type myrosinase gene was identified for the first time in horseradish. The gene termed ArMY2 was only expressed in young roots. A full-length cDNA encoding a myrosinase termed ArMy2 was isolated and heterologously expressed in Pichia pastoris. The recombinant His-tagged enzyme was characterized biochemically. Substrate affinity was 5 times higher towards gluconasturtiin than towards sinigrin. Gluconasturtiin was found to be the most abundant glucosinolate in young horseradish roots while sinigrin dominated in storage roots and leaves. This indicates that a specialized glucosinolate-myrosinase defense system might be active in young roots., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Plants can extensively transform contaminants after uptake through phase I and phase II metabolism to a large diversity of products. UPLC-QToF-MS was used to detect and identify metabolites of the bacteriostatic agent triclosan in a horseradish hairy root culture. Thirty-three metabolites of triclosan were recognized by a stepwise approach of mass defect filtering, multivariate data analysis, and isotope pattern filtering from a data set of several thousands of signals in the exposed culture. Structure proposals were elaborated for 23 triclosan metabolites on the basis of their MS data. The majority were identified as conjugates (phase II metabolites) such as saccharides or sulfosaccharides. Additionally, a disulfosaccharide was identified as a plant metabolite for the first time. Besides that, also conjugates of a phase I metabolite, hydroxytriclosan, were determined in horseradish tissue extracts. Dehalogenation products of triclosan were not observed. The large number of metabolites detected and identified in this study emphasizes the importance of a comprehensive analytical approach in studies on the uptake and fate of organic contaminants in plants.

Background: Horseradish plants (Armoracia rusticana) contain high concentrations of glucosinolates. Former studies have revealed that Armoracia plants cultivated in vitro have markedly lower glucosinolate concentrations than those grown in soils. Yet, these studies neglected that the sulfate concentration in the growth medium may have had a strong impact on glucosinolate metabolism. Accordingly, in this study horseradish in vitro plants were cultivated with differing sulfate concentrations and the glucosinolate concentrations were quantified by ion pair HPLC., Results: Cultivation in 1.7 mmol L(-1) sulfate (as used in the prior studies) resulted in the accumulation of 16.2 µmol g(-1) DW glucosinolates, while the glucosinolate concentration increased to more than 23 µmol g(-1) DW when 23.5 mmol L(-1) sulfate was used in the medium. Correspondingly, the glucosinolate concentration decreased to 1.6 µmol g(-1) DW when sulfate concentration was lowered to 0.2 mmol L(-1)., Conclusion: Since the glucosinolate accumulation in relation to the sulfate concentration follows a typical saturation curve, we deduce that the availability of sulfate determines the glucosinolate concentration in horseradish in vitro plants., (© 2012 Society of Chemical Industry.)

Pharmaceuticals from human or veterinary medication form a new class of micropollutants that poses a serious threat to our aquatic environment and its organisms. The intensively used nonsteroidal anti-inflammatory drug diclofenac is found in the environment worldwide due to its poor elimination during waste water treatment processes. In order to test phytoremediation as a tool for the removal of this drug from waste water, the uptake of the compound into plant tissues and its metabolic pathway was addressed using Hordeum vulgare (barley) and a hairy root cell culture of Armoracia rusticana (horse radish) as model species. Diclofenac is taken up by plants and undergoes rapid metabolization; already after 3h of exposure the drug and its metabolites could be detected in the plant tissues. Similar to its fate in mammalian cells the drug is activated in a phase I reaction resulting in the hydroxylated metabolite 4'OH-diclofenac which is conjugated subsequently in phase II to a glucopyranoside, a typical plant specific metabolite. After exposure to 10 and 100 μM diclofenac a concentration dependent formation of the hydroxylated metabolite was observed, while the formation of the phase II metabolite OH-diclofenac glucopyranoside was not positively affected by the higher concentration. To our knowledge this is the first time these two human painkiller metabolites are shown to occur in plant tissues., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Positive and negative effects of rare earth elements (REEs) in life have been reported in many papers, but the cellular mechanisms have not been answered, especially the action sites of REEs on plasma membrane are unknown. Proteins on/in the plasma membrane perform main functions of the plasma membrane. Cerium (Ce) is the richest REEs in crust. Thus, the interaction between Ce(III) and the proteins on/in the plasma membrane, the morphology of protoplast, and the contents of nutrient elements in protoplast of horseradish were investigated using the optimized combination of the fluorescence microscopy, fluorescence spectroscopy, circular dichroism, scanning electron microscopy, and X-ray energy dispersive spectroscopy. It was found that Ce(III) at the low concentrations (10, 30 μM) could interact with proteins on/in the plasma membrane of horseradish, leading to the improvement in the structure of membrane proteins and the plasma membrane, which accelerated the intra-/extra-cellular substance exchange and further promoted the development of cells. When horseradish was treated with Ce(III) at the high concentrations (60, 80 μM), Ce(III) also could interact with the proteins on/in the plasma membrane of horseradish, leading to the destruction in the structure of membrane proteins and the plasma membrane. These effects decelerated the intra-/extra-cellular substance exchange and further inhibited the development of cells. Thus, the interaction between Ce(III) and proteins on/in the plasma membrane in plants was an important reason of the positive and negative effects of Ce(III) on plants. The results would provide some references for understanding the cellular effect mechanisms of REEs on plants.

Lanthanum (La) is an important rare earth element in the ecological environment of plant. The proteins on the plasma membrane control the transport of molecules into and out of cell. It is very important to investigate the effect of La(III) on the proteins on the plasma membrane in the plant cell. In the present work, the interaction between La(III) and proteins on the plasma membrane of horseradish was investigated using optimization of the fluorescence microscopy and fluorescence spectroscopy. It is found that the fluorescence of the complex system of protoplasts and 1-aniline Kenai-8-sulfonic acid in horseradish treated with the low concentration of La(III) is increased compared with that of the control horseradish. The opposite effect is observed in horseradish treated with the high concentration of La(III). These results indicated that the low concentration of La(III) can interact with the proteins on the plasma membrane of horseradish, causing the improvement in the structure of proteins on the plasma membrane. The high concentration of La(III) can also interact with the proteins on the plasma membrane of horseradish, leading to the destruction of the structure of proteins on the plasma membrane. We demonstrate that the proteins on the plasma membrane are the targets of La(III) action on plant cell., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The wide application of rare earth elements (REEs) has led to the accumulation of REEs in soil and plant. Thereby, the effect of Tb(3+) on the contents of cytosolic nutrient elements in horseradish was investigated with the synchronous detection technique of scanning electron microscope and energy dispersive X-ray spectrometry. It was found for the first time that the foliar spraying treatment of Tb(3+) destroyed the structure of horseradish mesophyll cells, and then changed the contents of the cytosolic nutrient elements in horseradish, especially Ca. The effect of Tb(3+) was increased with increasing the concentration of Tb(3+). The hydroponical treatment of Tb(3+) could not obviously change the structure of protoplast and the contents of the cytosolic nutrient elements in horseradish leaves. The results indicated that the accumulation of Tb(3+) in soil and plant leaves displayed the different toxic effect on plant leaves., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

In order to understand the toxic mechanism of terbium ion (Tb(III)) on plants, the subcellular distribution of Tb(III) in horseradish, the effect of Tb(III) on the composition of the fatty acids in the cell membrane, the peroxidation of membrane lipid, the morphological character of protoplast, the cellular ultrastructure in horseradish were investigated using transmission electron microscopic autoradiography, molecular dynamics simulation, gas chromatography, scanning electron microscopy and transmission electron microscopy. The results show that Tb(III) could not enter the horseradish cell in the presence of 5 mgL(-1) Tb(III) and it was distributed on the cell wall and plasma membrane. The behavior caused the decrease in the contents of unsaturated fatty acids and then the increase in the peroxidation of membrane lipid. Thereby the structure of horseradish cell was damaged. The effects of Tb(III) mentioned above were aggravated in horseradish treated with 60 mgL(-1) Tb(III) because Tb(III) could enter the horseradish cell. It was a possible cytotoxic mechanism of Tb(III) on horseradish., (2010 Elsevier Ltd. All rights reserved.)

Background, Aim, and Scope: Pharmaceuticals and their metabolites are detected in the aquatic environment and our drinking water supplies. The need for high quality drinking water is one of the most challenging problems of our times, but still only little knowledge exists on the impact of these compounds on ecosystems, animals, and man. Biological waste water treatment in constructed wetlands is an effective and low-cost alternative, especially for the treatment of non-industrial, municipal waste water. In this situation, plants get in contact with pharmaceutical compounds and have to tackle their detoxification. The mechanisms for the detoxification of xenobiotics in plants are closely related to the mammalian system. An activation reaction (phase I) is followed by a conjugation (phase II) with hydrophilic molecules like glutathione or glucose. Phase III reactions can be summarized as storage, degradation, and transport of the xenobiotic conjugate. Until now, there is no information available on the fate of pharmaceuticals in plants. In this study, we want to investigate the fate and metabolism of N-acetyl-4-aminophenol (paracetamol) in plant tissues using the cell culture of Armoracia rusticana L. as a model system., Materials and Methods: A hairy root culture of A. rusticana was treated with acetaminophen in a liquid culture. The formation and identification of metabolites over time were analyzed using HPLC-DAD and LC-MSn techniques., Results: With LC-MS technique, we were able to detect paracetamol and identify three of its metabolites in root cells of A. rusticana. Six hours after incubation with 1 mM of acetaminophen, the distribution of acetaminophen and related metabolites in the cells resulted in 18% paracetamol, 64% paracetamol-glucoside, 17% paracetamol glutathione, and 1% of the corresponding cysteine conjugate., Discussion: The formation of two independently formed metabolites in plant root cells again revealed strong similarities between plant and mammalian detoxification systems. The detoxification mechanism of glucuronization in mammals is mirrored by glucosidation of xenobiotics in plants. Furthermore, in both systems, a glutathione conjugate is formed. Due to the existence of P450 enzymes in plants, the formation of the highly reactive NAPQI intermediate is possible., Conclusions: In this study, we introduce the hairy root cell culture of A. rusticana L. as a suitable model system to study the fate of acetaminophen in plant tissues. Our first results point to the direction of plants being able to take up and detoxify the model substrate paracetamol. These first findings underline the great potential of using plants for waste water treatments in constructed wetlands., Recommendations and Perspectives: This very first study on the detoxification of a widely used antipyretic agent in plant tissues again shows the flexibility of plant detoxification systems and their potential in waste water treatment facilities. This study covers only the very first steps of acetaminophen detoxification in plants; still, there is no data on long-term exposure as well as the possible impact of pharmaceuticals on the plant health and stress defense. Long-term experiments need to be performed to follow the fate of acetaminophen in root and leaf cells in a whole plant system, and to evaluate possible usage of plants for the remediation of acetaminophen from waste water.

The remodeling process of the arterial wall in atherosclerosis involves intimal thickening, which can be related to the barrier functions of the endothelial cell layer (ECL) and internal elastic lamina (IEL) using horseradish peroxidase (HRP) as a tracer. To evaluate the ECL and IEL permeabilities (PECL and PIEL, respectively) and intimal transport parameters, e.g., apparent HRP velocity (VI) and diffusivity, we compared simulations with a mathematical model to experimental data. In this study, we injected HRP into the vein of apolipoprotein E-null mice and measured HRP concentration profiles in lesioned areas of aortas. Lesion size was characterized by lower, middle, and upper ranges of the intimal/medial thickness (deltaI/deltaM): 01.0. The PECL (in micrometers per minute) of 5-mo-old mice in the middle range (0.98+/-0.14) was significantly greater than that in the lower range (0.21+/-0.03) but not significantly different from mice in the upper range (0.99+/-0.55). The PECL of 12-mo-old mice increased significantly with the relative intimal thickness: 0.27+/-0.04 in the lower range, 1.12+/-0.15 in the middle range, and 1.74+/-0.24 in the upper range. In both age groups, VI (in micrometers per minute) increased significantly from lower to upper ranges of intimal thickness. However, PIEL did not change significantly with relative intimal thickness and age. In the upper range of intimal thickness, PECL and VI were significantly greater in 12-mo-old mice than in 5-mo-old mice. These data indicate an interaction between lesion growth and aging that leads to progressive loss in the integrity of the endothelial barrier function. Furthermore, the IEL is not a significant barrier between the intima and tunica media in the atherosclerotic process.

The agricultural application of rare-earth elements (REEs) would promote REEs inevitably to enter in the environment and then to threaten the environmental safety and human health. Therefore, the distribution of the REEs ion, (141)Ce(III) and effects of La(III), Ce(III) and Tb(III) on the distribution of horseradish peroxidase (HRP) in horseradish mesophyll cells were investigated with electron microscopic radioautography and transmission electron microscopic cytochemistry. It was found for the first time that REEs ions can enter into the mesophyll cells, deposit in both extra and intra-cellular. Compared to the normal condition, after the horseradish leaves treated with La(III) or Tb(III), HRP located on the tonoplast is decreased and HRP is mainly located on the cell wall, while HRP is mainly located on the plasma membrane after the horseradish leaves were treated with Ce(III). This also indicated that REEs ions may regulate the plant growth through changing the distribution of enzymes.

Background and Aims: Rare earth elements (REEs) are used in agriculture and a large amount of them contaminate the environment and enter foods. The distribution and translocation of (141)Ce (III) in horseradish was investigated in order to help understand the biochemical behaviour and toxic mechanism of REEs in plants., Methods: The distribution and translocation of (141)Ce (III) in horseradish were investigated using autoradiography, liquid scintillation counting (LSC) and electron microscopic autoradiography (EMARG) techniques. The contents of (141)Ce (III) and nutrient elements were analysed using an inductively coupled plasma-atomic emission spectrometer (ICP-AES)., Results: The results from autoradiography and LSC indicated that (141)Ce (III) could be absorbed by horseradish and transferred from the leaf to the leaf-stalk and then to the root. The content of (141)Ce (III) in different parts of horseradish was as follows: root > leaf-stalk > leaf. The uptake rates of (141)Ce (III) in horseradish changed with the different organs and time. The content of (141)Ce (III) in developing leaves was greater than that in mature leaves. The results from EMARG indicated that (141)Ce (III) could penetrate through the cell membrane and enter the mesophyll cells, being present in both extra- and intra-cellular deposits. The contents of macronutrients in horseradish were decreased by (141)Ce (III) treatment., Conclusions: (141)Ce (III) can be absorbed and transferred between organs of horseradish with time, and the distribution was found to be different at different growth stages. (141)Ce (III) can enter the mesophyll cells via apoplast and symplast channels or via plasmodesmata. (141)Ce (III) can disturb the metabolism of macronutrients in horseradish.

Horseradish and soybean peroxidases (HRP and SBP, respectively) are useful biotechnological tools. HRP is often termed the classical plant heme peroxidase and although it has been studied for decades, our understanding has deepened since its cloning and subsequent expression, enabling numerous mutational and protein engineering studies. SBP, however, has been neglected until recently, despite offering a real alternative to HRP: SBP actually outperforms HRP in terms of stability and is now used in numerous biotechnological applications, including biosensors. Review of both is timely. This article summarizes and discusses the main insights into the structure and mechanism of HRP, with special emphasis on HRP mutagenesis, and outlines its use in a variety of applications. It also reviews the current knowledge and applications to date of SBP, particularly biosensors. The final paragraphs speculate on the future of plant heme-based peroxidases, with probable trends outlined and explored.

The type 1 copper sites of cupredoxins typically have a His(2)Cys equatorial ligand set with a weakly interacting axial Met, giving a distorted tetrahedral geometry. Natural variations to this coordination environment are known, and we have utilized paramagnetic (1)H NMR spectroscopy to study the active-site structure of umecyanin (UMC), a stellacyanin with an axial Gln ligand. The assigned spectra of the Cu(II) UMC and its Ni(II) derivative [Ni(II) UMC] demonstrate that this protein has the typical His(2)Cys equatorial coordination observed in other structurally characterized cupredoxins. The NMR spectrum of the Cu(II) protein does not exhibit any paramagnetically shifted resonances from the axial ligand, showing that this residue does not contribute to the singly occupied molecular orbital (SOMO) in Cu(II) UMC. The assigned paramagnetic (1)H NMR spectrum of Ni(II) UMC demonstrates that the axial Gln ligand coordinates in a monodentate fashion via its side-chain amide oxygen atom. The alkaline transition, a feature common to stellacyanins, influences all of the ligating residues but does not alter the coordination mode of the axial Gln ligand in UMC. The structural features which result in Cu(II) UMC possessing a classic type 1 site as compared to the perturbed type 1 center observed for other stellacyanins do not have a significant influence on the paramagnetic (1)H NMR spectra of the Cu(II) or Ni(II) proteins.

Many products of interest are synthesized in organized tissues, but not formed in suspension or callus culture. Therefore, most attention has been focused on root cultures. The transgenic plant,"hairy root", has brought us to dramatic improvements in growth rate and high content of desirable products. Since the roots are quite different from callus in morphology, the culture manner should be explored independently. By providing a growth environment, an elite hairy root can be a more attractive plant. Both of strain selection to generate more competent plants in breeding and engineering development are necessary to overcome various limitations. In this chapter the engineering issues involved in using hairy root culture are discussed, as follows. 1. Measurement of cell concentration on line, and a designing bioreactors for hairy root in liquid culture. 2. High cell density culture and its kinetic parameters. 3. Secretion of target products. 4. The micropropagation of the regenerated hairy root by means of artificial seed system. In some cases where callus and suspension culture show negligible productivity, organ culture will be necessary to achieve good formation. This study on hairy root culture indicates one of the best attempts to the recovery of products from the organ culture in plant biotechnology.

Remediation of soil and groundwater contaminated by leaking fuel storage tanks may be assisted by plants, although plant effects on abiotic and biotic removal processes remain unclear. The objectives of this study were to investigate abiotic and biotic removal of JP-8, a kerosene-based jet fuel, in soils with plants, and to determine the effects of plant-induced water movement. Loss of JP-8 in a dry-soil, control column was 25% after 5 months, primarily due to volatilization and gas-phase diffusion. By comparison, managed treatments with simulated surface spills averaged 86% mass reduction at 5 months, indicating an important contribution of biodegradation. Overall JP-8 mass reduction was similar in surface and subsurface-irrigated systems, indicating water content, not mode of water application, influences bioremediation in near-surface systems. The JP-8 concentration reductions in soil columns contaminated above a simulated watertable were 36% after 3 months and 50% after 12 months for vegetated columns compared to 26% and 34% in unplanted columns. Downward movement of JP-8 in unplanted columns was double that in planted columns. Near the groundwater table, JP-8 persists longer than near the soil surface. Plants promote upward movement of water and help draw spilled JP-8 to aerobic near-surface soil.