The tu8 mutation of Arabidopsis thaliana encoding a heterochromatin protein 1 homolog causes defects in the induction of secondary metabolite biosynthesis.

The tu8 mutant of Arabidopsis thaliana (L.) Heynh. was previously described as deficient in pathogen-induced auxin and glucosinolate (GSL) accumulation, as well as in heat-induced accumulation of cytosolic Hsp90, the latter feature was accom"panied by reduced thermotolerance at higher temperatures. The mutated gene was shown to be a novel allele of TERMINAL FLOWER2, encoding the only Arabidopsis homolog for heterochromatin protein 1 (Kim et al., 2004). In this report, we investigated the influence of heat stress on auxin and GSL content, as well as the accumulation of several secondary metabolites derived from the phenylpropanoid pathway, including anthocyanins and sinapine derivatives, in the mutant tu8. tu8 had less sinapine and sinapoyl esters compared to the wild type. In addition, the induction of sinapine by heat shock in Columbia was not found in tu8. Anthocyanins were also induced by heat stress in wild type plants, whereas tu8 showed only slight induction of these compounds and only at higher temperatures. GSLs were induced at higher temperatures in the wild type, but induction was absent in tu8. Transcript levels known to be involved in IAA/glucosinolate synthesis and metabolism (nitrilase and myrosinase) were examined and both showed developmental regulation, while only nitrilase mRNA levels differed between wild type and mutant seedlings. Treatment of Columbia and tu8 with jasmonic acid (JA), a known inducer of glucosinolates, showed differences between wild type and tu8 with respect to induction of individual GSLs and anthocyanins. However, the transcript level of the TU8/TFL2 gene after heat shock and jasmonate treatment did not change. Loss of function or altered function in the heterochromatin protein most likely lead to the pleiotropic phenotype observed for the tu8 mutant.

Absorption of kaempferol from endive, a source of kaempferol-3-glucuronide, in humans.

OBJECTIVE: To determine the absorption, excretion and metabolism of kaempferol in humans. DESIGN: A pharmacokinetic study of kaempferol from endive over 24 h. SUBJECTS: Four healthy males and four healthy females. RESULTS: Kaempferol, from a relatively low dose (9 mg), was absorbed from endive with a mean maximum plasma concentration of 0.1 microM, at a time of 5.8 h, indicating absorption from the distal section of the small intestine and/or the colon. Although a 7.5-fold interindividual variation between the highest and lowest maximum plasma concentration was observed, most individuals showed remarkably consistent pharmacokinetic profiles. This contrasts with profiles for other flavonoids that are absorbed predominantly from the large intestine (eg rutin). An average of 1.9% of the kaempferol dose was excreted in 24 h. Most subjects also showed an early absorption peak, probably corresponding to kaempferol-3-glucoside, present at a level of 14% in the endive. Kaempferol-3-glucuronide was the major compound detected in plasma and urine. Quercetin was not detected in plasma or urine indicating a lack of phase I hydroxylation of kaempferol. CONCLUSIONS: Kaempferol is absorbed more efficiently than quercetin in humans even at low oral doses. The predominant form in plasma is a 3-glucuronide conjugate, and interindividual variation in absorption and excretion is low, suggesting that urinary kaempferol could be used as a biomarker for exposure.

Hydrolysis by lactase phlorizin hydrolase is the first step in the uptake of daidzein glucosides by rat small intestine in vitro.

1. Isoflavones are naturally occurring oestrogenic compounds found in plants, where they exist in the glycosylated form. A proportion of ingested glycosides appears to be absorbed in the upper gastrointestinal tract, where enterocytes play an important role in their metabolism. 2. One hypothesis is that ingestion may involve hydrolysis by the luminally exposed enzyme lactase phlorizin hydrolase (LPH), an enzyme expressed specifically at the small intestinal brush border. 3. Using an everted sac preparation of rat jejunum and an inhibitor of LPH, we investigated the absorption of daidzein-O(7)-glucoside (daidzin) and the effect of LPH inhibition on this process. It was demonstrated that LPH plays a major role in the deglycosylation of daidzin. 4. The hydrolysis product, daidzein, is absorbed by epithelial cells and glucuronidated to daidzein-O(7)-glucuronide, which is subsequently exported primarily to the serosal (vascular) side of the tissue rather than to the luminal side. 5. A small but significant proportion of the intact glycoside is also transferred to the serosal compartment, and in the presence of an LPH inhibitor this was enhanced with a corresponding reduction in deglucosylation and glucuronidation. 6. The results indicate that that LPH plays an important role in the metabolism of glycosylated phytochemicals, and that the expression and activity of this enzyme in the small intestine can modify the profile of metabolites appearing in the circulation.

Rerouting the plant phenylpropanoid pathway by expression of a novel bacterial enoyl-CoA hydratase/lyase enzyme function.

The gene for a bacterial enoyl-CoA hydratase (crotonase) homolog (HCHL) previously shown to convert 4-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA to the corresponding hydroxybenzaldehydes in vitro provided an opportunity to subvert the plant phenylpropanoid pathway and channel carbon flux through 4-hydroxybenzaldehyde and the important flavor compound 4-hydroxy-3-methoxybenzaldehyde (vanillin). Expression of the Pseudomonas fluorescens AN103 HCHL gene in two generations of tobacco plants caused the development of phenotypic abnormalities, including stunting, interveinal chlorosis and senescence, curled leaf margins, low pollen production, and male sterility. In second generation progeny, the phenotype segregated with the transgene and transgenic siblings exhibited orange/red coloration of the vascular ring, distorted cells in the xylem and phloem bundles, and lignin modification/reduction. There was depletion of the principal phenolics concomitant with massive accumulation of novel metabolites, including the glucosides and glucose esters of 4-hydroxybenzoic acid and vanillic acid and the glucosides of 4-hydroxybenzyl alcohol and vanillyl alcohol. HCHL plants exhibited increased accumulation of transcripts for phenylalanine ammonia-lyase, cinnamate-4-hydroxylase, and 4-coumarate:CoA ligase, whereas beta-1,3-glucanase was suppressed. This study, exploiting the ability of a bacterial gene to divert plant secondary metabolism, provides insight into how plants modify inappropriately accumulated metabolites and reveals the consequences of depleting the major phenolic pools.

A cinnamoyl esterase from Aspergillus niger can break plant cell wall cross-links without release of free diferulic acids.

A cinnamoyl esterase, ferulic acid esterase A, from Aspergillus niger releases ferulic acid and 5-5- and 8-O-4-dehydrodiferulic acids from plant cell walls. The breakage of one or both ester bonds from dehydrodimer cross-links between plant cell wall polymers is essential for optimal action of carbohydrases on these substrates, but it is not known if cinnamoyl esterases can break these cross-links by cleaving one of the ester linkages which would not release the free dimer. It is difficult to determine the mechanism of the reaction on complex substrates, and so we have examined the catalytic properties of ferulic acid esterase A from Aspergillus niger using a range of synthetic ethyl esterified dehydrodimers (5-5-, 8-5-benzofuran and 8-O-4-) and two 5-5-diferulate oligosaccharides. Our results show that the esterase is able to cleave the three major dehydrodiferulate cross-links present in plant cell walls. The enzyme is highly specific at hydrolysing the 5-5- and the 8-5-benzofuran diferulates but the 8-O-4-is a poorer substrate. The hydrolysis of dehydrodiferulates to free acids occurs in two discrete steps, one involving dissociation of a monoesterified intermediate which is negatively charged at the pH of the reaction. Although ferulic acid esterase A was able to release monoesters as products of reactions with all three forms of diesters, only the 5-5- and the 8-O-4-monoesters were substrates for the enzyme, forming the corresponding free diferulic acids. The esterase cannot hydrolyse the second ester bond from the 8-5-benzofuran monoester and therefore, ferulic acid esterase A does not form 8-5-benzofuran diferulic acid. Therefore, ferulic acid esterase A from Aspergillus niger contributes to total plant cell wall degradation by cleaving at least one ester bond from the diferulate cross-links that exist between wall polymers but does not always release the free acid product.

Simultaneous time-varying systemic appearance of oral and hepatic glucose in adults monitored with stable isotopes.

The rates (and extent) of appearance of glucose in arterialized plasma from an oral glucose load and from liver (RaO, RaH) can be estimated in humans using radioisotopes, but estimates vary among laboratories. We investigated the use of stable isotopes and undertook 22 primed intravenous infusions of D-[6,6-2H2]glucose with an oral load including D-[13C6]glucose in healthy humans. The effective glucose pool volume (VS) had a lower limit of 230 ml/kg body weight (cf. 130 ml/kg commonly assumed). This VS in Steele's one-compartment model of glucose kinetics gave a systemic appearance from a 50-g oral glucose load per 70 kg body weight of 96 +/- 3% of that ingested, which compared with a theoretical value of approximately 95%. Mari's two-compartment model gave 100 +/- 3%. The two models gave practically identical RaO and RaH at each point in time and a plateau in the cumulative RaO when absorption was complete. Less than 3% of 13C was recycled to [13C3]glucose, suggesting that recycling errors were practically negligible in this study. Causes of variation among laboratories are identified. We conclude that stable isotopes provide a reliable and safe alternative to radioactive isotopes in these studies.

Identification of skatolyl hydroperoxide and its role in the peroxidase-catalysed oxidation of indol-3-yl acetic acid.

Indol-3-yl acetic acid (IAA, auxin) is a plant hormone whose degradation is a key determinant of plant growth and development. The first evidence for skatolyl hydroperoxide formation during the plant peroxidase-catalysed degradation of IAA has been obtained by electrospray MS. Skatolyl hydroperoxide degrades predominantly non-enzymically to oxindol-3-yl carbinol but in part enzymically into indol-3-yl methanol via a peroxidase cycle in which IAA acts as an electron donor. Skatolyl hydroperoxide is degradable by catalase. Horseradish peroxidase isoenzyme C (HRP-C) and anionic tobacco peroxidase (TOP) exhibit differences in their mechanisms of reaction. The insensitivity of the HRP-C-catalysed reaction to catalase is ascribed to the formation of HRP-C Compound III at the initiation step and its subsequent role in radical propagation. This is in contrast with the TOP-catalysed process in which skatolyl hydroperoxide has a key role. Indol-3-yl aldehyde is produced not via the peroxidase cycle but by catalysis involving ferrous peroxidase. Because indol-3-yl aldehyde is one of the main IAA-derived products identified in planta, we conclude that ferrous peroxidases participate in IAA catalytic transformations in vivo. A general scheme for peroxidase-catalysed IAA oxidation is presented.

Analysis of nisin A and some of its variants using Fourier transform ion cyclotron resonance mass spectrometry.

The lantibiotic nisin and some of its variants and degradation products have been characterized, using a 9.4-T Fourier transform ion cyclotron resonance mass spectrometer and electrospray ionization. The abundances of all products in the sample (i.e., major component, variants, degradation products, and adducts) have been measured quantitatively. The mass resolution obtained in the electrospray ionisation mass spectra was approximately 100,000 over the measured range. The resulting mass accuracy, better than 0.7 ppm (or within 0.001 Da) allowed the molecular masses and in many cases chemical formulae of most components in the mixture to be identified unambiguously. Additionally, amino acid sequence information on nisin and a variant [nisin + 18 Da] was obtained using sustained off-resonance irradiation collisional activated decomposition (SORI-CAD) of mass-selected precursor ions. Even after introducing collision gas into the mass analyser for the SORI-CAD experiments, the mass accuracy in the fragment ion mass spectra was approximately 5 ppm. It was established that the [nisin + 18 Da] molecule, present as a minor component in the mixture, was a species formed predominantly via hydration of nisin at position 33, i.e., [Ser33]nisin, with a small contribution due to hydration at position 5,[2-hydroxy-Ala5]nisin.

Stable isotope methods for studying nutrient mineral metabolism in humans.

Stable isotopes are safe, versatile, scientifically rigorous tools for studying nutrient metabolism in humans. They can be used to follow the metabolism of nutrient minerals from specific foods or diets, including the determination of efficiency of absorption and the effects of dietary constituents on absorption, and to investigate fundamental aspects of nutritional biochemistry. In this article we describe stable isotope methods and the results of some recent nutrient mineral metabolism studies in humans.

Comparison of high- and low-energy collision-induced dissociation tandem mass spectrometry in the analysis of glycoalkaloids and their aglycons.

Four aglycons (tomatidine, demissidine, solanidine, and solasodine) and three glycoalkaloids (α-tomatine, α-chaconine, and α-solanine) have been analyzed by positive ion liquid secondary ion high-energy and low-energy collision-induced dissociation (CID) tandem mass Spectrometry, performed on a four-sector (EBEB) and a hybrid (EBQQ) instrument, respectively. Both high- and low-energy collision-induced dissociation mass spectra of [M+H](+) ions of these compounds provided structural information that aided the characterization of the different aglycons and of the carbohydrate sequence and linkage sites in the glycoalkaloids. Low-energy CID favors charge-driven fragmentation of the aglycon rings, whilst high-energy CID spectra are more complex and contain additional ions that appear to result from charge-remote fragmentations, multiple cleavages, or complex charge-driven rearrangements. With respect to the structural characterization of the carbohydrate part, low-energy CID fragmentations of sugar residues in the glycoalkaloids generate Y n (+) ions and some low intensity Z n (+) ions; the high-energy spectra also exhibit strong (1,5)X n (+) ions, formed by multiple cleavage of the sugar ring, and significant Z n (+) ions.

Mass spectrometric methods for studying nutrient mineral and trace element absorption and metabolism in humans using stable isotopes. A review.

Mass spectrometric methods for determining stable isotopes of nutrient minerals and trace elements in human metabolic studies are described and discussed. The advantages and disadvantages of the techniques of electron ionization, fast atom bombardment, thermal ionization, and inductively coupled plasma and gas chromatography mass spectrometry are evaluated with reference to their accuracy, precision, sensitivity, and convenience, and the demands of human nutrition research. Examples of specific applications are described and the significance of current developments in mass spectrometry are discussed with reference to present and probable future research needs.

Analysis of glycoalkaloids from potato shoots and tomatoes by four-sector tandem mass spectrometry with scanning-array detection: comparison of positive ion and negative ion methods.

A wide range of glycoalkaloids from potato shoots and tomatoes, including trisaccharide-containing glycoalkaloids (alpha-chaconine, alpha-solanine, and alpha-solasonine), tetrasaccharide-containing glycoalkaloids (alpha-tomatine and demissine), and disaccharide-containing glycoalkaloids (beta 1-chaconine, beta 2-chaconine, and beta-solamargine), have been studied by both positive and negative ion liquid secondary ion and four-sector tandem mass spectrometry with scanning-array detection. In positive ion mode, collisionally induced dissociation tandem mass spectra of the [M + H]+ ions induce three major fragmentation processes, Z cleavage, Y cleavage, and 1,5X cleavage, which are structurally informative. Signals resulting from Z0, Y0, and 1,5X0 cleavages provide information on the nature of various aglycone moieties in all glycoalkaloids. Linkages and positions of the sugars in trisaccharide- and tetrasaccharide-containing glycoalkaloids are indicated by the presence or absence of the ions corresponding to Z alpha/beta and Y alpha/beta cleavages and intensity differences of the peaks due to 1,5X alpha and 1,5X beta cleavages, respectively. In negative ion mode, collisionally induced dissociation tandem mass spectra of the [M - H]- ions induce Y cleavage as the major fragmentation process. The location of the terminal sugars in branched trisaccharide and tetrasaccharide glycoalkaloids is indicated by the difference in intensity of the ions due to Y alpha cleavage and Y beta cleavage. Isomeric structures cannot, however, be differentiated unambiguously; complete structural assignment is only possible by NMR of purified components. Both positive and negative ion tandem mass spectrometry are considered to be suitable for the characterisation of glycoalkaloids in mixtures. The positive ion method has the advantage of (i) a lower detection limit than in conventional mass spectrometry; (ii) numerous and intense fragment ions which are structurally informative; and (iii) the capability of analyzing minor components in crude extracts. Comparable analysis by other analytical means would not have provided the amount of structural information on the components in the glycoalkaloid mixtures.

Comparison of fast-atom bombardment, thermal ionization and inductively coupled plasma mass spectrometry for the measurement of 64Zn/67Zn stable isotopes in human nutrition studies.

Fast-atom bombardment, thermal ionization and inductively coupled plasma mass spectrometry are contrasted in comparative measurements of 64Zn/67Zn isotope ratios on samples isolated in the course of a human mineral-nutrition experiment. The data are evaluated with reference to the precision normally required in enriched stable-isotope mineral-nutrition studies, and the convenience of the mass spectrometric techniques is also compared.

Thermospray liquid chromatography-mass spectrometry in food and agricultural research.

A thermospray liquid chromatography-mass spectrometry interface connected to a double-focusing magnetic sector instrument has been applied successfully to the solution of a number of analytical problems in food and agricultural research. Examples of the qualitative analysis of some anti-nutrients, plant growth control hormones, biotechnology products and a plant polyphenol are presented and discussed.