Myrosinase Compatible Simultaneous Determination of Glucosinolates and Allyl Isothiocyanate by Capillary Electrophoresis Micellar Electrokinetic Chromatography (CE-MEKC).

INTRODUCTION: The functional food Cruciferous vegetables contain glucosinolates which are decomposed by the myrosinase enzyme upon tissue damage. The isothiocyanates are the most frequent decomposition products. Because of their various bioactivities, these compounds and the myrosinase is of high interest to many scientific fields. OBJECTIVE: Development of a capillary electrophoresis method capable of myrosinase-compatible, simultaneous quantification of glucosinolates and isothiocyanates. METHODS: Capillary electrochromatography parameters were optimised, followed by optimisation of a myrosinase-compatible derivatisation procedure for isothiocyanates. Vegetable extracts (Brussels sprouts, horseradish, radish and watercress) were tested for myrosinase activity, glucosinolate content and isothiocyanate conversion rate. Allyl isothiocyanate was quantified in some food products. RESULTS: The method allows quantification of sinigrin, gluonasturtiin and allyl isothiocyanate after myrosinase compatible derivatisation in-vial by mercaptoacetic acid. The chromatograhpic separation takes 2.5 min (short-end injection) or 15 min (long-end injection). For the tested vegetables, measured myrosinase activity was between 0.960-27.694 and 0.461-26.322 µmol/min/mg protein, glucosinolate content was between 0-2291.8 and 0-248.5 µg/g fresh weight for sinigrin and gluconastrutiin, respectively. The possible specificity of plants to different glucosinolates was also shown. Allyl isothiocyanate release rate was different in different vegetables (73.13 - 102.13%). The method could also be used for quantification of allyl isothiocyanate from food products. CONCLUSIONS: The presented capillary electrophoresis method requires a minimal amount of sample and contains only a few sample preparation steps, and can be used in several applications (glucosinolate determination, myrosinase activity measurement, isothiocyanate release estimation). Copyright © 2016 John Wiley & Sons, Ltd.

Biodegradation of C.I. Acid Blue 92 by Nasturtium officinale: Study of Some Physiological Responses and Metabolic Fate of Dye.

The present study was conducted to evaluate the potential of aquatic vascular plant, Nasturtium officinale, for degradation of C.I. Acid Blue 92 (AB92). The effect of operational parameters such as initial dye concentration, plant biomass, pH, and temperature on the efficiency of biological decolorization process was determined. The reusability of the plant in long term repetitive operations confirmed the biological degradation process. The by-products formed during biodegradation process were identified by GC-MS technique. The effects of the dye on several plant physiological responses such as photosynthetic pigments content and antioxidant enzymes activity were investigated. The content of chlorophyll and carotenoids was significantly reduced at 20 mg/L of the dye. The activities of superoxide dismutase and peroxidase were remarkably increased in the plant root verifying their importance in plant tolerance to the dye contamination.

Nickel accumulation and its effect on biomass, protein content and antioxidative enzymes in roots and leaves of watercress (Nasturtium officinale R. Br.).

In order to understand its response towards nickel stress, watercress (Nasturtium officinale R. Br.) was exposed to nickel (1-25 mg/L) for 1, 3, 5 and 7 days. The accumulation and translocation of nickel were determined and the influence of nickel on biomass, protein content and enzymatic antioxidants was examined for both roots and leaves. It was determined that N. officinale could accumulate appreciable amounts of Ni in both roots and leaves. Nickel accumulated particularly in the roots of plants. Biomass increased at low nickel concentrations but certain measurable change was not found at high concentrations. Under stress conditions the antioxidant enzymes were up-regulated compared to control. An increase in protein content and enzyme activities was observed at moderate exposure conditions followed by a decline at both roots and leaves. The maximum enzyme activities were observed at different exposure conditions. Our results showed that N. officinale had the capacity to overcome nickel-induced stress especially at moderate nickel exposure. Therefore, N. officinale may be used as a phytoremediator in moderately polluted aquatic ecosystems.

Sequential injection lab-on-valve system for the determination of the activity of peroxidase in vegetables.

Horseradish peroxidase (HRP) has been broadly used and investigated for many analytical purposes; it is an enzyme that catalyzes the reduction of hydrogen peroxide in the presence of a reducing compound. The objective of this work was to develop a methodology for the spectrophotometric determination of the activity of peroxidase in vegetable extracts using a flow method with a sequential injection lab-on-valve format. The developed system is based on the reaction between hydrogen peroxide (H(2)O(2)) and 2,2-azinobis(3-ethylbenzothiazoline-6)sulfonic acid (ABTS) catalyzed by the enzyme (HRP). The method presented a sample consumption of 15 microL per assay and a consumption of ABTS and H(2)O(2) of 24 microg and 12 microg per assay, respectively. It was also possible to monitor online the thermal inactivation of peroxidase at different temperature ranges.

Effects of lead on the activities of antioxidant enzymes in watercress, Nasturtium officinale R. Br.

The aim of the present study is to evaluate the oxidative effects of lead with increased concentrations by the determination of antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and ascorbate peroxidase (AP)) and lipid peroxidation levels in the stem and leaves of watercress (Nasturtium officinale R. Br.) which was exposed to lead acetate, Pb (CH3COOH)2 regime with concentrations of 0, 50, 100, 200, 250, and 500 mg/L Pb in a hydroponic culture. After 14 days, accumulation of lipid peroxidation in stems and leaves and changes in activity of antioxidant enzymes were determined spectrophotometrically. The maximum accumulation was observed in the highest concentration group. In this group, lipid peroxidation levels were three times higher than the control group in the stem and leaves. The highest induction in SOD and GR activities were determined at 200 mg/L Pb group in stem, whereas CAT and AP activities were higher than other groups at the concentration of 250 and 100 mg/L Pb, respectively. The increase in CAT activity was found to be greater than GR, SOD, and AP activities in stems of watercress under Pb treatment. Both lead accumulation and antioxidant enzyme responses were higher in stems than in leaves. The results of the present study suggested that the induction in antioxidant responses could be occurring as an adaptive mechanism to the oxidative potential of lead accumulation.

A real-time fluorogenic assay for the visualization of glycoside hydrolase activity in planta.

There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e.g. protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin beta-glycoside of a xylogluco-oligosaccharide (XXXG-beta-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pK(a) (5.8) and large extinction coefficient (epsilon 62,000 M(-1) cm(-1)), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-beta-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-beta-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.

Transformation of Nasturtium officinale, Barbarea verna and Arabis caucasica for hairy roots and glucosinolate-myrosinase system production.

Hairy roots of Nasturtium officinale, Barbarea verna and Arabis caucasica with active glucosinolate-myrosinase system were obtained after transformation with Agrobacterium rhizogenes. Hairy roots of N. officinale produced phenylalanine-derived gluconasturtiin and glucotropaeolin (max. 24 and 7 mg g(-1) DW). B. verna and A. caucasica hairy roots produced gluconasturtiin (max. 41 mg g(-1) DW) and methionine-derived glucoiberverin (max. 32 mg g(-1) DW), respectively. Treatment of the roots with amino acid precursors of glucosinolate or/and cysteine biosynthesis increased levels of glucosinolate production, combinations of phenylalanine with cysteine (for gluconasturtiin and glucotropaeolin) and methionine with o-acetylserine (for glucoiberverin) were the most effective.

Analysis of nasturtium TmNXG1 complexes by crystallography and molecular dynamics provides detailed insight into substrate recognition by family GH16 xyloglucan endo-transglycosylases and endo-hydrolases.

Reorganization and degradation of the wall crosslinking and seed storage polysaccharide xyloglucan by glycoside hydrolase family 16 (GH16) endo-transglycosylases and hydrolases are crucial to the growth of the majority of land plants, affecting processes as diverse as germination, morphogenesis, and fruit ripening. A high-resolution, three-dimensional structure of a nasturtium (Tropaeolum majus) endo-xyloglucanase loop mutant, TmNXG1-DeltaYNIIG, with an oligosaccharide product bound in the negative active-site subsites, has been solved by X-ray crystallography. Comparison of this novel complex to that of the strict xyloglucan endo-transglycosylase PttXET16-34 from hybrid aspen (Populus tremula x tremuloides), previously solved with a xylogluco-oligosaccharide bound in the positive subsites, highlighted key protein structures that affect the disparate catalytic activities displayed by these closely related enzymes. Combination of these "partial" active-site complexes through molecular dynamics simulations in water allowed modeling of wild-type TmNXG1, TmNXG1-DeltaYNIIG, and wild-type PttXET16-34 in complex with a xyloglucan octadecasaccharide spanning the entire catalytic cleft. A comprehensive analysis of these full-length complexes underscored the importance of various loops lining the active site. Subtle differences leading to a tighter hydrogen bonding pattern on the negative (glycosyl donor) binding subsites, together with loop flexibility on the positive (glycosyl acceptor) binding subsites appear to favor hydrolysis over transglycosylation in GH16 xyloglucan-active enzymes.

Seed-specific heterologous expression of a nasturtium FAE gene in Arabidopsis results in a dramatic increase in the proportion of erucic acid.

The fatty acid elongase [often designated FAE or beta-(or 3-) ketoacyl-CoA synthase] is a condensing enzyme and is the first component of the elongation complex involved in synthesis of erucic acid (22:1) in seeds of garden nasturtium (Tropaeolum majus). Using a degenerate primers approach, a cDNA of a putative embryo FAE was obtained showing high homology to known plant elongases. This cDNA contains a 1,512-bp open reading frame that encodes a protein of 504 amino acids. A genomic clone of the nasturtium FAE was isolated and sequence analyses indicated the absence of introns. Northern hybridization showed the expression of this nasturtium FAE gene to be restricted to the embryo. Southern hybridization revealed the nasturtium beta-ketoacyl-CoA synthase to be encoded by a small multigene family. To establish the function of the elongase homolog, the cDNA was introduced into two different heterologous chromosomal backgrounds (Arabidopsis and tobacco [Nicotiana tabacum]) under the control of a seed-specific (napin) promoter and the tandem 35S promoter, respectively. Seed-specific expression resulted in up to an 8-fold increase in erucic acid proportions in Arabidopsis seed oil, while constitutive expression in transgenic tobacco tissue resulted in increased proportions of very long chain saturated fatty acids. These results indicate that the nasturtium FAE gene encodes a condensing enzyme involved in the biosynthesis of very long chain fatty acids, utilizing monounsaturated and saturated acyl substrates. Given its strong and unique preference for elongating 20:1-CoA, the utility of the FAE gene product for directing or engineering increased synthesis of erucic acid is discussed.