In situ monitoring of the shikimate pathway: a combinatorial approach of Raman reverse stable isotope probing and hyperspectral imaging.

Sensing and visualization of metabolites and metabolic pathways in situ are significant requirements for tracking their spatiotemporal dynamics in a non-destructive manner. The shikimate pathway is an important cellular mechanism that leads to the de novo synthesis of many compounds containing aromatic rings of high importance such as phenylalanine, tyrosine, and tryptophan. In this work, we present a cost-effective and extraction-free method based on the principles of stable isotope-coupled Raman spectroscopy and hyperspectral Raman imaging to monitor and visualize the activity of the shikimate pathway. We also demonstrated the applicability of this approach for nascent aromatic amino acid localization and tracking turnover dynamics in both prokaryotic and eukaryotic model systems. This method can emerge as a promising tool for both qualitative and semi-quantitative in situ metabolomics, contributing to a better understanding of aromatic ring-containing metabolite dynamics across various organisms.

Effects of direct-fed microbial supplement on ruminal and plasma metabolome of early-lactation dairy cows: Untargeted metabolomics approach.

We examined the effects of 2 multispecies direct-fed microbial (DFM) supplements on ruminal and plasma metabolome of early-lactation dairy cows using a high-coverage untargeted metabolomics approach. A total of 45 multiparous Holstein cows (41 ± 7 DIM) were enrolled for the 14-d pre-experimental and 91-d experimental period and were a subset from a lactation performance study, which used 114 cows. Cows were blocked using pre-experimental energy-corrected milk yield and randomly assigned within each block to 1 of 3 treatments: (1) corn silage-based diet with no DFM supplement (control; CON), (2) basal diet top-dressed with a mixture of Lactobacillus animalis and Propionibacterium freudenreichii at 3 × 109 cfu/d (PRO-A), or (3) basal diet top-dressed with a mixture of L. animalis, P. freudenreichii, Bacillus subtilis, and Bacillus licheniformis at 11.8 × 109 cfu/d (PRO-B). The basal diet was fed ad libitum daily as a TMR at 0600 and 1200 h for a duration of 91 d. Rumen fluid and blood samples were taken on d -3, 28, 49, 70, and 91 and immediately stored at -80°C. Before analysis, ruminal and plasma samples from d 28, 49, 70, and 91 were composited. An in-depth, untargeted metabolome profile of the composite rumen and plasma samples and the d -3 samples was developed by using a chemical isotope labeling/liquid chromatography-mass spectrometry (LC-MS)-based technique. Differentially abundant metabolites (taking into account fold change [FC] values and false discovery rates [FDR]) were identified with a volcano plot. In the rumen, compared with the CON diet, supplemental PRO-A increased (FC ≥1.2; FDR ≤0.05) the relative concentrations of 9 metabolites, including 2-hydroxy-2,4-pentadienoic acid, glutaric acid, quinolinic acid, and shikimic acid, and PRO-B increased relative concentrations of 16 metabolites, including 2-hydroxy-2,4-pentadienoic acid, glutaric acid, 16-hydroxypalmitic acid, and 2 propionate precursors (succinic and methylsuccinic acids). Relative to PRO-A, supplemental PRO-B increased (FC ≥1.2; FDR ≤0.05) relative rumen concentrations of 3 metabolites, 16-hydroxypalmitic acid, indole-3-carboxylic acid, and 5-aminopentanoic acid, but reduced relative rumen concentrations of 13 metabolites, including carnitine, threonic acid, and shikimic acid. Compared with the CON diet, relative concentrations of 13 plasma metabolites, including myxochelin A and glyceraldehyde, were increased (FC ≥1.2; FDR ≤0.05) by PRO-A supplementation, whereas those of 9 plasma metabolites, including 4-(2-aminophenyl)-2,4-dioxobutanoic acid, N-acetylornithine, and S-norlaudanosolin, were reduced (FC ≤0.83; FDR ≤0.05). Supplemental PRO-B increased (FC ≥1.2; FDR ≤0.05) relative concentrations of 9 plasma metabolites, including trans-o-hydroxybenzylidenepyruvic acid and 3-methylsalicylaldehyde, and reduced relative concentrations of 4 plasma metabolites, including ß-ethynylserine and kynurenine. Pathway analysis of the differentially abundant metabolites in both rumen and plasma revealed that these metabolites are involved in AA and fatty acid metabolism and have antimicrobial and immune-stimulating properties. The results of this study demonstrated that dietary supplementation with either PRO-A or PRO-B altered the plasma and ruminal metabolome. Notably, ruminal and plasma metabolites involved in the metabolism of AA and fatty acids and those with immunomodulatory properties were altered by either or both of the 2 microbial additives.

Assessment of fresh star anise (Illicium verum Hook.f.) drying methods for influencing drying characteristics, color, flavor, volatile oil and shikimic acid.

In order to evaluate the effects of drying methods on the drying characteristics, quality (color, volatile oil (VO) content, shikimic acid (SA) content, trans-anethole content in the star anise volatile oil (TA-O)) and flavor components of star anise (Illicium verum Hook.f.), we tested five different methods (hot air drying (HAD), heat pump drying (HPD), far infrared radiation drying (FIRD), microwave drying (MD), and sun drying (SD)) with or without blanching to dry fresh star anise. Results showed MD had a shorter drying time than others, as well as the highest SA content (125.56 mg/g d.b.). HPD sample exhibited higher VO content (12.27% d.b.) and TA-O (113.30 mg/g d.b.) than those dried with other methods. HPD can improved the dominant flavor compounds of star anise, including trans-anethole (4165.46 mg/100 g d.b.), estragole (176.50 mg/100 g d.b.), linalool (280.69 mg/100 g d.b.), and (+)-limonene (471.18 mg/100 g d.b.). Samples treated with HPD-B had the highest comprehensive score (4.59) in the flavor principal component analysis. Therefore, HPD was more suitable for star anise drying as it maintaining quality. The better quality (higher flavor quality and better appearance) was found in HPD-B.

Mass spectrometry characterization, antioxidant activity, and cytotoxicity of the peel and pulp extracts of Pitomba.

The fruit of the Talisia esculenta tree, is largely consumed and appreciated for its bittersweet taste; however, detailed information on its constituent bioactive compounds is still scarce. Therefore, this study aims to screen the antioxidant activity by six methods and determine the chemical profile of the pitomba fruit peel and pulp by electrospray ionization-Fourier transform-mass spectrometry. This is the first study attempting to identify the bioactive compounds in the pitomba fruit peel. Consequently, 19 and 14 compounds were identified in the ethanolic and hexanic peel extracts, while 7 and 10 compounds were detected in the ethanolic and hexanic pulp extracts, respectively. The common compounds across the board were citric acid, ascorbic acid, and shikimic acid. In addition, the ethanolic peel extract exhibited a high 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (54.21-81.41%). The obtained results highlight the importance the pitomba fruit as a promising source of natural compounds with high antioxidant activities.

Effects of pre-harvest glyphosate use on protein composition and shikimic acid accumulation in spring wheat.

During wheat cultivation, glyphosate-based herbicides are recommended to be applied a week prior to harvest during the ripe stage of physiological maturity. However, some grains may not be at this physiological stage due to non-uniform maturation within the field. The goal of this study was to determine the effect of glyphosate-based herbicide timing on the chemistry of wheat gluten proteins and shikimic acid accumulation. The results of the study indicate that pre-harvest glyphosate application does not impact the amino acid composition, protein secondary structure or gluten protein composition. However, pre-harvest glyphosate application decreased the molecular weight of SDS extractable and unextractable proteins, and significantly increased the amount of shikimic acid accumulation, especially when applied early. Thus, this study indicates that pre-harvest use of glyphosate-based herbicides can cause significant differences in wheat protein chemistry and shikimic acid levels, especially when applied earlier than recommended, emphasizing the importance of timely application.

Physiological performance of glyphosate and imazamox mixtures on Amaranthus palmeri sensitive and resistant to glyphosate.

The herbicides glyphosate and imazamox inhibit the biosynthetic pathway of aromatic amino acids (AAA) and branched-chain amino acids (BCAA), respectively. Both herbicides share several physiological effects in the processes triggered in plants after herbicide application that kills the plant, and mixtures of both herbicides are being used. The aim of this study was to evaluate the physiological effects in the mixture of glyphosate and imazamox in glyphosate-sensitive (GS) and -resistant (GR) populations of the troublesome weed Amaranthus palmeri. The changes detected in the physiological parameters after herbicide mixtures application were similar and even less to the changes detected after individual treatments. This pattern was detected in shikimate, amino acid and carbohydrate content, and it was independent of the EPSPS copy number, as it was detected in both populations. In the case of the transcriptional pattern of the AAA pathway after glyphosate, interesting and contrary interactions with imazamox treatment were detected for both populations; enhancement of the effect in the GS population and alleviation in the GR population. At the transcriptional level, no cross regulation between AAA and BCAA inhibitors was confirmed. This study suggests that mixtures are equally or less toxic than herbicides alone, and would implicate careful considerations when applying the herbicide mixtures.

Effect of sodium nitroprusside treatment on shikimate and phenylpropanoid pathways of apple fruit.

Blue mould caused by Penicillium expansum is one of the important diseases of apple fruit during storage. Phenylpropanoid pathway is an important induction mechanism that can utilize downstream metabolites of shikimate pathway to synthesize a series of secondary metabolites. Apple fruit (cv. Fuji) were treated with sodium nitroprusside (SNP) to study its effect on blue mould, shikimate and phenylpropanoid pathways. The results showed that 1.0 mmol L-1 SNP significantly inhibited lesion development of apple fruit inoculated with P. expansum. The results also indicated that SNP enhanced MdDHQS, MdSKDH, MdSK and MdEPSPS genes expressions, increased shikimic acid, tryptophan, tyrosine and phenylalanine contents in apple fruit. The activities of phenylalanine ammonialyase, 4-coumarate: coenzyme A, ligase, cinnamate 4-hydroxylase, lignin, total phenolic compounds and flavonoids contents in apple fruit were also increased by SNP treatment. These results suggest that SNP might modulate shikimate and phenylpropanoid pathways to enhance disease resistance of apple fruit.

Bioactive compounds content and antimicrobial activities of wild edible Asteraceae species of the Mediterranean flora under commercial cultivation conditions.

Nine wild edible species belonging to Astreaceae family, native to the Mediterranean basin were tested for their chemical composition (phenolic compounds, tocopherols, and organic acids) and antimicrobial activities over two growing periods, apart from Scolymus hispanicus and Hedypnois cretica which were tested for only one growing period. Flavonoids were the most abundant phenolic compounds in all the species, except for the case of Taraxacum species where significant amounts of chicoric acid were detected, while phenolic compounds content increased in the 2nd growing period by 4.6-397.4% for the tested species. α- and ß-tocopherols were the main tocopherols, apart from Taraxacum sp. where significant amounts of γ-and δ-tocopherols (18.32 and 16.31 µg/100 g fresh weight) were detected, while total tocopherols content either increased (Reicardia picroides, Picris echioides, Urospermum picroides, and Taraxacum officinale) or decreased (Hymenonema graecum, Sonchus oleraceus, Taraxacum sp.) in the 2nd growing period. Oxalic acid was the most abundant organic acid, with the highest content (972 mg/100 g fresh weight) being observed in H. graecum (L.) DC. in the 1st growing period. Moreover, with the exception of H. graecum and S. olearaceus, total organic acids content increased in the 2nd growing period. Significant antimicrobial activities were observed against Bacillus cereus, Salmonella typhimurium and Penicillium ochrochloron for all the studied species. In conclusion, the studied species showed great potential for commercial cultivation, while plant extracts could find use in the food industry as alternative food preservatives.

Recombinant AroL-Catalyzed Phosphorylation for the Efficient Synthesis of Shikimic Acid 3-Phosphate.

Shikimic acid 3-phosphate, as a central metabolite of the shikimate pathway, is of high interest as enzyme substrate for 5-enolpyruvoyl-shikimate 3-phosphate synthase, a drug target in infectious diseases and a prime enzyme target for the herbicide glyphosate. As the important substrate shikimic acid 3-phosphate is only accessible via a chemical multi-step route, a new straightforward preparative one-step enzymatic phosphorylation of shikimate using a stable recombinant shikimate kinase has been developed for the selective phosphorylation of shikimate in the 3-position. Highly active shikimate kinase is produced by straightforward expression of a synthetic aroL gene in Escherichia coli. The time course of the shikimate kinase-catalyzed phosphorylation is investigated by 1 H- and 31 P-NMR, using the phosphoenolpyruvate/pyruvate kinase system for the regeneration of the ATP cofactor. This enables the development of a quantitative biocatalytic 3-phosphorylation of shikimic acid. After a standard workup procedure, a good yield of shikimic acid 3-phosphate, with high HPLC- and NMR purity, is obtained. This efficient biocatalytic synthesis of shikimic acid 3-phosphate is superior to any other method and has been successfully scaled up to multi-gram scale.

Novel comprehensive multidimensional liquid chromatography approach for elucidation of the microbosphere of shikimate-producing Escherichia coli SP1.1/pKD15.071 strain.

Shikimic acid is a intermediate of aromatic amino acid biosynthesis and the preferred starting material for production of the most commonly prescribed anti-influenza drug, Tamiflu. Its six-membered carbocyclic ring is adorned with several chiral centers and various functionalities, making shikimic acid a valuable chiral synthon. When microbially-produced, in addition to shikimic acid, numerous other metabolites are exported out of the cytoplasm and accumulate in the culture medium. This extracellular matrix of metabolites is referred to as the microbosphere. Due to the high sample complexity, in this study, the microbosphere of shikimate-producing Escherichia coli SP1.1/pKD15.071 was analyzed by liquid chromatography and comprehensive two-dimensional liquid chromatography coupled to photodiode array and mass spectrometry detection. GC analysis of the trimethylsilyl derivatives was also carried out in order to support the elucidation of the selected metabolites in the microbosphere. The elucidation of the metabolic fraction of this bacterial strain might be of valid aid for improving, through genetic changes, the concentration and yield of shikimic acid synthesized from glucose. Graphical abstract.

Quantitation of Compounds in Wine Using (1)H NMR Spectroscopy: Description of the Method and Collaborative Study.

To examine whether NMR analysis is a suitable method for the quantitative determination of wine components, an international collaborative trial was organized to evaluate the method according to the international regulations and guidelines of the German Institute for Standardization/International Organization for Standardization, AOAC INTERNATIONAL, the International Union of Pure and Applied Chemistry, and the International Organization of Vine and Wine. Sugars such as glucose; acids such as malic, acetic, fumaric, and shikimic acids (the latter two as minor components); and sorbic acid, a preservative, were selected for the exemplary quantitative determination of substances in wine. Selection criteria for the examination of sample material included different NMR spectral signal types (singlet and multiplet), as well as the suitability of the proposed substances for manual integration at different levels of challenge (e.g., interference as a result of the necessary suppression of a water signal or the coverage of different typical wine concentration ranges for a selection of major components, minor components, and additives). To show that this method can be universally applied, NMR measurement and the method of evaluation were not strictly elucidated. Fifteen international laboratories participated in the collaborative trial and determined six parameters in 10 samples. The values, in particular the reproducibility SD (SR), were compared with the expected Horwitz SD (SH) by forming the quotient SR/SH (i.e., the HorRat value). The resulting HorRat values of most parameters were predominantly between 0.6 and 1.5, and thus of an acceptable range.

Simultaneous determination of shikimic acid, salicylic acid and jasmonic acid in wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses.

The presence and relative concentration of phytohormones may be regarded as a good indicator of an organism's physiological state. The integration of the rolC gene from Agrobacterium rhizogenes and of the rat glucocorticoid receptor (gr) in Nicotiana langsdorffii Weinmann plants has shown to determine various physiological and metabolic effects. The analysis of wild and transgenic N. langsdorffii plants, exposed to different abiotic stresses (high temperature, water deficit, and high chromium concentrations) was conducted, in order to investigate the metabolic effects of the inserted genes in response to the applied stresses. The development of a new analytical procedure was necessary, in order to assure the simultaneous determination of analytes and to obtain an adequately low limit of quantification. For the first time, a sensitive HPLC-HRMS quantitative method for the simultaneous determination of salicylic acid, jasmonic acid and shikimic acid was developed and validated. The method was applied to 80 plant samples, permitting the evaluation of plant stress responses and highlighting some metabolic mechanisms. Salicylic, jasmonic and shikimic acids proved to be suitable for the comprehension of plant stress responses. Chemical and heat stresses showed to induce the highest changes in plant hormonal status, differently affecting plant response. The potential of each genetic modification toward the applied stresses was marked and particularly the resistance of the gr modified plants was evidenced. This work provides new information in the study of N. langsdorffii and transgenic organisms, which could be useful for the further application of these transgenes.

Metabolite profiling in 18 Saudi date palm fruit cultivars and their antioxidant potential via UPLC-qTOF-MS and multivariate data analyses.

Date palm fruit (Phoenix dactylifera) is not only one of the most economically significant plants in the Middle East, but also valued for its nutritional impact, and for which development of analytical methods is ongoing to help distinguish its many cultivars. This study attempts to characterize the primary and secondary metabolite profiles of 18 date cultivars from Saudi Arabia. A total of 44 metabolites extracted from the fruit peel were evaluated in a UPLC-qTOF-MS based metabolomics analysis including flavonoids, phenolic acids and fatty acids. The predominant flavones were glycosides of luteolin and chrysoeriol, as well as quercetin conjugates, whereas caffeoyl shikimic acid was the main hydroxycinnamic acid conjugate. GC-MS was further utilized to identify the primary metabolites in fruits (i.e. sugars) with glucose and fructose accounting for up to 95% of TIC among most cultivars. PCA and OPLS analyses revealed that flavone versus flavonol distribution in fruit were the main contributors for cultivar segregation. The antioxidant activity of date fruit samples was correlated with their total phenolics as determined by DPPH and CUPRAC assays. Dkheni Saudi and Shalabi Madina cultivars, appearing as the most distant in clustering analyses exhibited the strongest antioxidant effect suggesting that multivariate data analysis could help determine which date cultivars ought to be prioritized for future agricultural development.

[Production suitability regionalization study of Pinus massoniana].

The distribution, yield and sample information data of Pinus massoniana was obtained by document literature and sample investigation. Based on sample data from 12 provinces including 414 sample plots and environment factors in China,the distribution regionalization of P. massoniana was predicted by using Maxent and spatial analysis function of ArcGIS. The results showed that the northernmost distribution of P. massoniana was 33.5 degrees north latitude, and it mainly distributed in the southeast in China. Based on plant age, plant height, yield per plant and other growth index from 414 sample plots, combined vegetation form and other data, the growth regionalization of P. massoniana was carried out by using SPSS and related functions of ArcGIS. The results showed that Fujian, Guizhou and Guangxi had a lager distribution area of P. massoniana, meanwhile, it had a relatively higher yield of fresh pine needles. The relational model between environmental factors and shikimic acid,and procyanidin, and the total lignans was constructed by using SPSS regression analysis method. Then the spatial calculation function of ArcGIS was used tocarry out the quality regionalization of P. massoniana based on the relational model. The results showed that east of Sichuan, Guizhou, Chongqing had a good pine needles quality. Based on the distribution, growth and quality regionalization, the production suitability regionalization of P. massoniana was carried out. The results showed that the optimal planting base region mainly distributed in east of Sichuan, middle and east of Guizhou, and east of Guangxi.

Metabolic Profiling and Enzyme Analyses Indicate a Potential Role of Antioxidant Systems in Complementing Glyphosate Resistance in an Amaranthus palmeri Biotype.

Metabolomics and biochemical assays were employed to identify physiological perturbations induced by a commercial formulation of glyphosate in susceptible (S) and resistant (R) biotypes of Amaranthus palmeri. At 8 h after treatment (HAT), compared to the respective water-treated control, cellular metabolism of both biotypes were similarly perturbed by glyphosate, resulting in abundance of most metabolites including shikimic acid, amino acids, organic acids and sugars. However, by 80 HAT the metabolite pool of glyphosate-treated R-biotype was similar to that of the control S- and R-biotypes, indicating a potential physiological recovery. Furthermore, the glyphosate-treated R-biotype had lower reactive oxygen species (ROS) damage, higher ROS scavenging activity, and higher levels of potential antioxidant compounds derived from the phenylpropanoid pathway. Thus, metabolomics, in conjunction with biochemical assays, indicate that glyphosate-induced metabolic perturbations are not limited to the shikimate pathway, and the oxidant quenching efficiency could potentially complement the glyphosate resistance in this R-biotype.

Development of an ESI-LC-MS-based assay for kinetic evaluation of Mycobacterium tuberculosis shikimate kinase activity and inhibition.

A simple and reliable liquid chromatography-mass spectrometry (LC-MS) assay has been developed and validated for the kinetic characterization and evaluation of inhibitors of shikimate kinase from Mycobacterium tuberculosis (MtSK), a potential target for the development of novel antitubercular drugs. This assay is based on the direct determination of the reaction product shikimate-3-phosphate (S3P) using electrospray ionization (ESI) and a quadrupole time-of-flight (Q-TOF) detector. A comparative analysis of the kinetic parameters of MtSK obtained by the LC-MS assay with those obtained by a conventional UV-assay was performed. Kinetic parameters determined by LC-MS were in excellent agreement with those obtained from the UV assay, demonstrating the accuracy, and reliability of this method. The validated assay was successfully applied to the kinetic characterization of a known inhibitor of shikimate kinase; inhibition constants and mode of inhibition were accurately delineated with LC-MS.

Glyphosate spray drift in Coffea arabica - sensitivity of coffee plants and possible use of shikimic acid as a biomarker for glyphosate exposure.

Glyphosate is widely used in coffee plantations to control weeds. Lacking selectivity, glyphosate spray drift is suspected to cause adverse effects in coffee plants. Symptoms caused by glyphosate can be similar to those produced by other stress factors. However, shikimic acid accumulation should be a useful biomarker for glyphosate exposure as shown for other crops. The aim of this study was to assess the sensitivity of coffee plants towards glyphosate on different biological response variables and to evaluate the use of shikimic acid as biomarker. Dose-response experiments yielded ED50 values (50% effect dose) in the range of 38-550 ga.e.ha(-1) depending on the quantitative or qualitative variable monitored. The frequency of plants showing symptoms was the most sensitive variable. The best sampling time for shikimic acid accumulation was 1-2 weeks after glyphosate application, depending on experimental conditions. The highest shikimic acid accumulation was observed in young leaves. Shikimic acid is a suitable biomarker for a glyphosate exposure in coffee, using only young leaves for the analysis. Young coffee plants are susceptible to glyphosate damage. If symptoms are absent the risk of severe crop damage or yield loss is low.

Phenolic compounds of apple cultivars resistant or susceptible to Venturia inaequalis.

CONTENT: Phenolic compounds play an important role in the plant defense mechanism and are responsible for antioxidant capacity in fruits and vegetables. It is known that the phenolics can determine in the leaves of plants which are resistant/susceptible to fungal infections. OBJECTIVE: This study investigated the total phenolic compounds, content of shikimic acid from 33 different apple cultivars leaves infected with Venturia inaequalis [(Cke). Wint.] cultured in Fruit Research Station, in Egirdir, Isparta, Turkey. MATERIALS AND METHODS: Leaves of apple cultivars were collected three times in an interval of 30 d from July to September in 2010, and analyzed using HPLC methods to detect changes in the amount of the phenolic compounds and shikimic acid. RESULTS: Total phenolic compounds and shikimic acid in resistant/moderate susceptible apple cultivars were higher than susceptible apple cultuvars, although not statistically different between resistant and susceptible apples. The content of shikimic acid was statistically higher only in the leaves of the domestic cultivar Ankara güzeli on all three dates. DISCUSSION AND CONCLUSION: Recently, there have been increased studies trying to explain the resistance mechanism in plants. Natural resistance genes are investigated in some apple cultivars and new resistance varieties which have resistant genes are identified daily. Our study hold to determine the relationship between the phenolic compounds and the expression of resistance seems to be promising.

Metabolomic analysis of the polyphenols in germinating mung beans (Vigna radiata) seeds and sprouts.

BACKGROUND: The mung bean (Vigna radiata) is a key food crop in much of Asia and contains plentiful biological activities to prevent human disease. Mung bean sprouts have more plentiful metabolites and activities after germination. RESULTS: The metabolite profile of polyphenols in the germination process was described using the methods of ultra-high-performance liquid chromatography coupled with time-of-flight mass spectrometry and partial least squares discriminant analysis. Sprouts from different periods were clearly discriminated from each other. Eight flavonoids - vitexin, isovitexin, rutin, kaempferol 3-O-rutinoside, isoquercitrin, genistein, daidzein and isorhamnetin - and two phenolic acids - shikimic acid and caffeic acid - were thought to be chemical markers of the sprouts. The method of high-performance liquid chromatography with diode array detection was established to quantitatively analyze the eight chemical markers of flavonoids, and provides good linearity, repeatability, intra- and inter-day precision, accuracy and recovery. The main metabolic and transformation pathways of the polyphenols in the germination process were discussed. CONCLUSION: The proposed method is sensitive, rapid and robust. Understanding the complete profile of polyphenol metabolites in the germination process may be useful for better utilizing mung beans sprouts as the raw materials of functional food, health products and cosmetics.

Response to metal stress of Nicotiana langsdorffii plants wild-type and transgenic for the rat glucocorticoid receptor gene.

Recently our findings have shown that the integration of the gene coding for the rat gluco-corticoid receptor (GR receptor) in Nicotiana langsdorffii plants induced morphophysiological effects in transgenic plants through the modification of their hormonal pattern. Phytohormones play a key role in plant responses to many different biotic and abiotic stresses since a modified hormonal profile up-regulates the activation of secondary metabolites involved in the response to stress. In this work transgenic GR plants and isogenic wild type genotypes were exposed to metal stress by treating them with 30ppm cadmium(II) or 50ppm chromium(VI). Hormonal patterns along with changes in key response related metabolites were then monitored and compared. Heavy metal up-take was found to be lower in the GR plants. The transgenic plants exhibited higher values of S-abscisic acid (S-ABA) and 3-indole acetic acid (IAA), salicylic acid and total polyphenols, chlorogenic acid and antiradical activity, compared to the untransformed wild type plants. Both Cd and Cr treatments led to an increase in hormone concentrations and secondary metabolites only in wild type plants. Analysis of the results suggests that the stress responses due to changes in the plant's hormonal system may derive from the interaction between the GR receptor and phytosteroids, which are known to play a key role in plant physiology and development.