Effects of selenium on antioxidant enzyme activity and bioaccessibility of arsenic in arsenic-stressed radish.

Consuming arsenic (As)-contaminated vegetables is the main route of As exposure in humans. The present study focused on the alterations in antioxidant enzymatic activities and As bioaccessibility in As-contaminated radish subjected to Se. Compared to the CK group, the total As content in raw radish was reduced by 27.5 ± 1.3%, and the bioaccessibility of As was reduced by 21.9 ± 2.3% in the 6 mg Se kg-1 treatment group. The total As content in the treatment groups decreased first but then increased with increasing Se application in raw radish, gastric (G) fraction and gastrointestinal (GI) fraction, while the antioxidant activity exhibited the opposite trend. The results revealed that a low amount of Se effectively blocks the accumulation of As in radish, improves the antioxidant activity in radish and reduces the bioaccessibility of As. These findings provide new ideas for effectively alleviating the spread of As to the human body through the food chain.

Genome-wide analysis of purple acid phosphatase structure and expression in ten vegetable species.

BACKGROUND: Acquisition of external phosphorus (P) and optimisation of internal P are essential for plant growth and development, and insufficient availability of P in soils is a major challenge in agriculture. Members of the purple acid phosphatase (PAP) family of enzymes are candidates for increasing P use efficiency. Herein, we identified PAP homologs in the genomes of 10 vegetable species, along with Arabidopsis thaliana and Amborella trichopoda as references, to provide fundamental knowledge for this family. RESULTS: Phylogenetic analysis of protein sequences revealed nine distinct clades, indicating that functional differentiation of extant PAPs was established prior to the emergence of early angiosperms, and conserved among homologs in each clade. Analysis of transcript abundance in different tissues (root, stem, leaf, flower, and fruit) and following phosphates (Pi) starvation treatments from published RNA-seq transcriptome datasets facilitated comprehensive evaluation of expression patterns, and some groups of tissue-specific and Pi starvation-induced PAPs were characterised. Conserved motifs identified from upstream sequences of homologs that are highly expressed in particular tissues or following starvation treatment suggests that divergence in PAP gene expression is associated with cis-acting elements in promoters. CONCLUSIONS: The genome-wide analysis of PAP enzyme structure and transcriptional expression patterns advance our understanding of PAP family in vegetables genomes. Therefore, PAP homologs with known enzyme structures and expression profiles could serve as targets for plant breeding and/or genetic engineering programs to improve P acquisition and use.

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.

Quality-related enzymes in fruit and vegetable products: effects of novel food processing technologies, part 1: high-pressure processing.

The activity of endogenous deteriorative enzymes together with microbial growth (with associated enzymatic activity) and/or other non-enzymatic (usually oxidative) reactions considerably shorten the shelf life of fruits and vegetable products. Thermal processing is commonly used by the food industry for enzyme and microbial inactivation and is generally effective in this regard. However, thermal processing may cause undesirable changes in product's sensory as well as nutritional attributes. Over the last 20 years, there has been a great deal of interest shown by both the food industry and academia in exploring alternative food processing technologies that use minimal heat and/or preservatives. One of the technologies that have been investigated in this context is high-pressure processing (HPP). This review deals with HPP focusing on its effectiveness for controlling quality-degrading enzymes in horticultural products. The scientific literature on the effects of HPP on plant enzymes, mechanism of action, and intrinsic and extrinsic factors that influence the effectiveness of HPP for controlling plant enzymes is critically reviewed. HPP inactivates vegetative microbial cells at ambient temperature conditions, resulting in a very high retention of the nutritional and sensory characteristics of the fresh product. Enzymes such as polyphenol oxidase (PPO), peroxidase (POD), and pectin methylesterase (PME) are highly resistant to HPP and are at most partially inactivated under commercially feasible conditions, although their sensitivity towards pressure depends on their origin as well as their environment. Polygalacturonase (PG) and lipoxygenase (LOX) on the other hand are relatively more pressure sensitive and can be substantially inactivated by HPP at commercially feasible conditions. The retention and activation of enzymes such as PME by HPP can be beneficially used for improving the texture and other quality attributes of processed horticultural products as well as for creating novel structures that are not feasible with thermal processing.

Novel self-compatible lines of Brassica rapa L. isolated from the Japanese bulk-populations.

Self-incompatibility (SI) in Brassicaceae is sporophytically controlled by a single S-locus with multi allelic variety. The male S determinant, SP11/SCR (S-locus protein 11/S-locus cysteine-rich protein), is a small cysteine-rich protein, and the female S determinant, SRK (S-locus receptor kinase), functions as a receptor for SP11 at the surface of stigma papilla cells. Although a few of the following downstream factors in the SP11-SRK signaling cascade have been identified, a comprehensive understanding of the SI mechanism still remains unexplained in Brassicaceae. Analysis of self-compatible (SC) mutants is significant for understanding the molecular mechanism in SI reactions, thus we screened SC lines from a variety of Japanese bulk-populations of B. rapa vegetables. Two lines, TSC4 and TSC28, seem to have disruptions in the SI signaling cascade, while the other line, TSC2, seems to have a deficiency in a female S determinant, SRK. In TSC4 and TSC28, known SI-related factors, i.e. SRK, SP11, MLPK (M-locus protein kinase), THL (thioredoxin-h-like), and ARC1 (arm repeat containing 1), were expressed normally, and their expression levels were comparable with those in SI lines. On a B. rapa genetic linkage map, potential SC genes in TSC4 and TSC28 were mapped on linkage groups A3 and A1, respectively, whereas MLPK, ARC1, and THL were mapped on A3, A4, and A6, respectively. Although potential SC genes of TSC4 and MLPK were on the same linkage group, their positions were apparently independent. These results indicate that the SC genes of TSC4 and TSC28 are independent from the S-locus or known SI-related genes. Thus, the SC lines selected here have mutations in novel factors of the SI signaling cascade, and they will contribute to fill pieces in a signal transduction pathway of the SI system in Brassicaceae.

Stability of hydroperoxide lyase activity from Amaranthus tricolor (Amaranthus mangostanus L.) leaves: influence of selected additives.

BACKGROUND: Hydroperoxide lyase (HPL) has potential value for the flavour additive industry. Currently, the production and application of HPL suffer from stability problems. The objective of this study was to investigate the stabilisation of HPL preparation from Amaranthus tricolor leaves by the addition of selected chemical additives. RESULTS: Amaranthus tricolor leaves were identified as a particularly rich source of 13-HPL activity. The addition of 100 g L(-1) sucrose and trehalose to microsomal HPL prior to lyophilisation could retain nearly 100% enzymatic activity, compared to only 20% for the lyophilised control. The lyophilised microsomal HPL containing sucrose maintained full activity for even 40 days storage at -20 degrees C. For HPL solution, glycerol was effective for long-term stability at -20 degrees C. Moreover, poyols (sucrose and trehalose) and amino acid (glycine) enhanced the thermostability of HPL, while KCl and polyol mannitol decreased the thermostability of HPL. CONCLUSION: The flavour-producing enzyme HPL, found in the leaves of Amaranthus tricolor, was stabilised by the addition of chemical additives.

Map-based cloning of a protein kinase gene conferring disease resistance in tomato.

The Pto gene in tomato confers resistance to races of Pseudomonas syringae pv. tomato that carry the avirulence gene avrPto. A yeast artificial chromosome clone that spans the Pto region was identified and used to probe a leaf complementary DNA (cDNA) library. A cDNA clone was isolated that represents a gene family, at least six members of which genetically cosegregate with Pto. When susceptible tomato plants were transformed with a cDNA from this family, they were resistant to the pathogen. Analysis of the amino acid sequence revealed similarity to serine-threonine protein kinases, suggesting a role for Pto in a signal transduction pathway.

Fine monitoring of major phenolic compounds in lettuce and escarole leaves during storage.

Two varieties of lettuce (Lactuca sativa L. var. crispa and capitata) and one variety of escarole (Cichorium endivia var. latifolium) were chosen due to their different browning sensitivity during storage as minimally processed products. The changes in the compositions and contents of the primary polyphenolic compounds were investigated during the first few hours of storage and then after 1, 2, 3, and 6 days of storage at 6°C and revealed sharp variations. Browning development, activity of phenylalanine ammonia lyase, and concentration of ascorbic acid were also examined during storage. The content of chicoric acid, which was the most abundant phenolic compound, varied substantially during the first 24 hr of storage and between the different varieties. Oak leaf lettuce, which was the most sensitive variety to browning, was characterized by a higher maintained chicoric acid level with a constant decreased chlorogenic acid level during the storage period. PRACTICAL APPLICATIONS: Browning development is responsible for the short shelf life of minimally processed vegetables, such as lettuce (Lactuca sativa). Polyphenolic compounds, as substrates of enzymatic reactions, are involved in the browning susceptibility of leaves. Comparisons of the profiles and contents of these compounds in different leaves showed dramatic variations during storage. This study provides additional information to better control, optimize minimally processed produce and select more suitable leaves for the fresh-cut industry.

Characterization of mammalian sedoheptulokinase and mechanism of formation of erythritol in sedoheptulokinase deficiency.

Our aim was to identify the product formed by sedoheptulokinase and to understand the mechanism of formation of erythritol in patients with sedoheptulokinase deficiency. Mouse recombinant sedoheptulokinase was found to be virtually specific for sedoheptulose and its reaction product was identified as sedoheptulose 7-phosphate. Assays of sedoheptulose in plant extracts disclosed that this sugar is present in carrots ( approximately 7mumol/g) and in several fruits. Sedoheptulose 1-phosphate is shown to be a substrate for aldolase B, which cleaves it to dihydroxyacetone-phosphate and erythrose. This suggests that, in patients deficient in sedoheptulose-7-kinase, sedoheptulose is phosphorylated by fructokinase to sedoheptulose 1-phosphate. Cleavage of the latter by aldolase B would lead to the formation of erythrose, which would then be reduced to erythritol.

Heavy metal accumulation, risk assessment and integrated biomarker responses of local vegetables: A case study along the Le'an river.

In this research, Ganzhou Chinese Cabbage (Brassica rapa pekinensis), Native Purple Garlic (Allium sativum L) and Leping Radish (Raphanus sativus L) widely planted and distributed along the Le'an River were chosen in the present study. Soil physical-chemical properties, nutrients contents as well as heavy metals elements accumulated in both soils and vegetables collected from 24 sites were analyzed by lab analysis combined with statistical method which was also used for calculation of contamination factor, pollution indexes and hazardous index. Heavy metals accumulation in soils were revealed with higher level, and copper and cadmium exceeded the background values by 8.82 and 16.73 times on average, which were also significantly related with the distribution of nonferrous metal processing enterprises. Heavy metal elements accumulated in vegetables were fully consistent with the finding of pollution characteristics in soils. Peroxidase biomarkers in vegetables, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), reduced glutathione (GSH) and lipoperoxidation (as TBARS), as well as integrated biomarker responses (IBR) were determined to give a reliable response after exposing of contaminants. Heavy metals accumulation ability and biomarker responses for three vegetables were usually determined in the following decrease trend: Ganzhou Chinese Cabbage > Native Purple Garlic > Leping Radish. Compared with peroxidase biomarkers activities or contents of control site, all the measured biomarkers in polluted sites showed significantly responses, indicating potential relationship between pollutants stresses and biomarker responses. This study also revealed that the IBR values were coordinated well with the pollutants concentrations.

Secondary structure and conformational change of mushroom polyphenol oxidase during thermosonication treatment by using FTIR spectroscopy.

To understand the conformational changes of mushroom PPO, the secondary structural change of the enzyme during thermosonication treatment at different power (60, 80 and 100%), temperature (20-60°C) and time (0-30min) combinations was investigated by using FTIR spectroscopy and compared with the change in enzyme activity. The enzyme inactivation higher than 99% was obtained at 100% amplitude at 60°C for 10min. FTIR studies showed that marked spectral changes were noted after ultrasound treatment at 20°C. The α-helix and ß-sheet contents decreased, while aggregated ß-sheet, turns and random coil contents increased as temperature increased up to 60°C during thermosonication treatment for 10min indicating protein denaturation. Aggregated bands located at 1683 and 1616cm(-1) became evident after ultrasound treatment at 40°C. When temperature was lowered back to 25°C, from ultrasound treatment at 60°C, these bands were still observed, indicating the irreversible change in the structure.

Analysis of the structure, substrate specificity, and mechanism of squash glycerol-3-phosphate (1)-acyltransferase.

BACKGROUND: Glycerol-3-phosphate (1)-acyltransferase(G3PAT) catalyzes the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acyl-CoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol-3-phosphate. G3PATs can either be selective, preferentially using the unsaturated fatty acid, oleate (C18:1), as the acyl donor, or nonselective, using either oleate or the saturated fatty acid, palmitate (C16:0), at comparable rates. The differential substrate specificity for saturated versus unsaturated fatty acids seen within this enzyme family has been implicated in the sensitivity of plants to chilling temperatures. RESULTS: The three-dimensional structure of recombinant G3PAT from squash chloroplast has been determined to 1.9 A resolution by X-ray crystallography using the technique of multiple isomorphous replacement and provides the first representative structure of an enzyme of this class. CONCLUSIONS: The tertiary structure of G3PAT comprises two domains, the larger of which, domain II, features an extensive cleft lined by hydrophobic residues and contains at one end a cluster of positively charged residues flanked by a H(X)(4)D motif, which is conserved amongst many glycerolipid acyltransferases. We predict that these hydrophobic and positively charged residues represent the binding sites for the fatty acyl substrate and the phosphate moiety of the glycerol 3-phosphate, respectively, and that the H(X)(4)D motif is a critical component of the enzyme's catalytic machinery.

Comparative analysis on the distribution of protease activities among fruits and vegetable resources.

In this study, a comparative analysis on the distribution of protease activities among 90 plant resources, including fruits and vegetables, has been performed. Protease activities of plant extracts were assayed at different pH values (pH 3.0, pH 7.5 and pH 10.5) using casein as a substrate. Ten fruits and thirteen vegetables show protease activities above 10U/g. Pineapple, fig and papaya, which are used for commercial protease production, exhibited high protease activities. Additionally, high protease activities were detected in kiwifruit (28.8U/g), broccoli (16.9U/g), ginger (16.6U/g), leek (32.7U/g) and red pepper (15.8U/g) at different pH values. SDS-PAGE and zymograms confirmed that various types of proteases existed in the five plant extracts and might be explored. Furthermore, five plant extracts were treated by different protease inhibitors. These results show that there are still many plant resources unexplored, which may be promising candidates for plant-derived protease production.

Cloning and sequencing of the gene encoding spinach ferredoxin-dependent glutamate synthase.

The nucleotide sequences of two cDNA clones, totalling 4948 bp in length, encoding 98% of the 1483 amino acids of the mature form of the ferredoxin-dependent glutamate synthase of spinach chloroplasts have been determined. The amino-terminal sequence of the enzyme has been determined by direct sequencing of the protein. The deduced amino-acid sequence of the spinach enzyme is 83% identical to that of the ferredoxin-dependent maize enzyme and shows significant sequence homology to two prokaryotic NAD(P)H-dependent glutamate synthases. Analysis of spinach genomic DNA indicates the presence of a single-copy gene for the spinach enzyme. Northern analysis reveals the presence of a single 5.5 kb transcript, which is present in higher levels in young spinach leaves than in older leaves.

The type 2 copper of ascorbate oxidase.

Ascorbate oxidase, dissolved in Hepes or sodium phosphate buffers, was analyzed by EPR and activity measurements before and after storage at -30 degrees C and 77 K. The specific activity was somewhat higher in the phosphate buffer, about 3500-3700 Dawson units compared to about 3100 units of the enzyme dissolved in Hepes buffer. After storage at -30 degrees C the activity fell to 1400-2000 units in the phosphate buffer but only to 2600-2800 units in the Hepes buffer. Large changes occurred in the EPR spectrum of enzyme dissolved in the phosphate buffer after storing at -30 degrees C suggesting an alteration of the type 2 copper site. These changes were, however, reverted when the samples were thawed and rapidly frozen at 77 K. Copper analysis showed that about 50% of the total copper was EPR detected. The type 2 Cu2+ EPR intensity was in most samples close to 25% of the total EPR intensity. This low contribution of type 2 Cu2+ could not be changed if the enzyme was completely reduced and reoxidized, treated with Fe(CN)6(3), large excess of NaF, addition of 50% (v/v) ethylene glycol or dialyzed against 0.1 M Mes buffer (pH 5.5). Since the crystal structure shows that there are one each of types 1 and 2 copper in the monomers there must be another species with an EPR signal rather different from these two copper species. This signal is proposed to originate from some trinuclear centers. The EPR simulations show that it is possible to house a broad unresolved signal under the resolved type 1 and 2 signals so that the total integral becomes 50% of the total copper in the molecule.

Characterization of a cDNA encoding cottonseed catalase.

A 1.7 kb cDNA clone was isolated from our lambda gt11 library constructed from poly(A) RNA of 24-h-old cotyledons. The cDNA encodes a full-length catalase peptide (492 amino acid residues). The calculated molecular mass is 56,800, similar to that determined for purified enzyme (57,000 SDS-PAGE). Among higher plant catalases, this cotton catalase shows the highest amino acid sequence identity (85%) to the subunit of homotetrameric maize CAT 1, a developmental counterpart to the homotetrameric CAT A isoform of cotton seeds. Comparison of sequences from cotton, sweet potato, maize CAT 1, and yeast with bovine catalase revealed that the amino acid residues and regions that are involved in catalytic activity and/or required to maintain basic catalase structure, are highly conserved. The C-terminus region, which has the lowest nucleotide sequence identity between plant and mammalian catalases, does not terminate with a tripeptide, S-K/R/H-L, a putative targeting signal for peroxisomal proteins.

A spectrometric study of the Co(2+)-activated ribulose-1,5-bisphosphate carboxylase reaction during turnover and at different pH.

EPR as well as optical absorption studies of the Co(2+)-activated ribulose-1,5-bisphosphate carboxylase/oxygenase under turnover conditions show that the formation of the two detectable intermediates are pH dependent. The amount of one of them, which earlier has been proposed to be a metal coordinated endiol of ribulose-1,5-bisphosphate (Brändén et al. (1987) Biochim. Biophys. Acta 916, 298-303), increased with increasing pH. Distinct optical absorption bands could be assigned to this intermediate and a pH profile could be made. It is therefore proposed that a base with a pKa value of about 8 is responsible for the enzyme-catalysed abstraction of a proton from ribulose-1,5-bisphosphate in order to form the metal coordinated endiol of ribulose-1,5-bisphosphate.

Crystallization and X-ray analysis of a multienzyme complex containing RUBISCO and RuBP.

Single crystals of a multienzyme complex isolated from spinach leaves, and containing RUBISCO bound to the substrate RuBP have been grown and characterized. The crystals belong to the orthorhombic space group P2(1)2(1)2 with a = 173 A, b = 134 A and c = 112 A, and contain two enzyme complex molecules in the unit cell. Diffraction data to 2.5 A resolution have been collected on the sychrotron source at the photon factory in Japan. Initial structure determination has been carried out using the molecular replacement method. The RUBISCO molecule in the complex has the normal L8S8 subunit configuration, and difference electron density is clearly observed for the other component enzymes and the RuBP substrate.