System-Wide Hypersensitive Response-Associated Transcriptome and Metabolome Reprogramming in Tomato
Etalo, D.W. ; Stulemeijer, I.J.E. ; Esse, H.P. van; Vos, R.C.H. de; Bouwmeester, H.J. ; Joosten, M.H.A.J. - \ 2013
Plant Physiology 162 (2013)3. - ISSN 0032-0889 - p. 1599 - 1617.
programmed cell-death - pathogen pseudomonas-syringae - campestris pv. vesicatoria - glutathione s-transferases - amino-acid catabolism - leaf rust resistance - higher-plant cells - mass-spectrometry - cladosporium-fulvum - functional-analysis
The hypersensitive response (HR) is considered to be the hallmark of the resistance response of plants to pathogens. To study HR-associated transcriptome and metabolome reprogramming in tomato (Solanum lycopersicum), we used plants that express both a resistance gene to Cladosporium fulvum and the matching avirulence gene of this pathogen. In these plants, massive reprogramming occurred, and we found that the HR and associated processes are highly energy demanding. Ubiquitin-dependent protein degradation, hydrolysis of sugars, and lipid catabolism are used as alternative sources of amino acids, energy, and carbon skeletons, respectively. We observed strong accumulation of secondary metabolites, such as hydroxycinnamic acid amides. Coregulated expression of WRKY transcription factors and genes known to be involved in the HR, in addition to a strong enrichment of the W-box WRKY-binding motif in the promoter sequences of the coregulated genes, point to WRKYs as the most prominent orchestrators of the HR. Our study has revealed several novel HR-related genes, and reverse genetics tools will allow us to understand the role of each individual component in the HR.
Reconstitution of the Costunolide Biosynthetic Pathway in Yeast and Nicotiana benthamiana.
Liu, Q. ; Majdi, M. ; Cankar, K. ; Goedbloed, M.A. ; Charnikhova, T. ; Verstappen, F.W.A. ; Vos, R.C.H. de; Beekwilder, M.J. ; Krol, S. van der; Bouwmeester, H.J. - \ 2011
PLoS ONE 6 (2011)8. - ISSN 1932-6203 - 12 p.
glutathione s-transferases - germacrene-a synthase - nf-kappa-b - sesquiterpene lactones - antimalarial-drug - mass-spectrometry - expression - arabidopsis - cloning - chicory
The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g-1 FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.
Comparison of two ecotypes of the metal hyperaccumulator Thlaspi caerulescens (J. & C. PRESL) at the transcriptional level
Plessl, M. ; Rigola, D. ; Hassinen, V.H. ; Tervahauta, A. ; Karenlampi, S. ; Schat, H. ; Aarts, M.G.M. ; Ernst, D. - \ 2010
Protoplasma 239 (2010)1-4. - ISSN 0033-183X - p. 81 - 93.
glutathione s-transferases - arabidopsis-thaliana - nicotianamine synthase - nitrate reductase - oxidative stress - plasma-membrane - gene-expression - nitric-oxide - 1-phosphate synthase - phaseolus-vulgaris
This paper investigates differences in gene expression among the two Thlaspi caerulescens ecotypes La Calamine (LC) and Lellingen (LE) that have been shown to differ in metal tolerance and metal uptake. LC originates from a metalliferous soil and tolerates higher metal concentrations than LE which originates from a non-metalliferous soil. The two ecotypes were treated with different levels of zinc in solution culture, and differences in gene expression were assessed through application of a cDNA microarray consisting of 1,700 root and 2,700 shoot cDNAs. Hybridisation of root and shoot cDNA from the two ecotypes revealed a total of 257 differentially expressed genes. The regulation of selected genes was verified by quantitative reverse transcriptase polymerase chain reaction. Comparison of the expression profiles of the two ecotypes suggests that LC has a higher capacity to cope with reactive oxygen species and to avoid the formation of peroxynitrite. Furthermore, increased transcripts for the genes encoding for water channel proteins could explain the higher Zn tolerance of LC compared to LE. The higher Zn tolerance of LC was reflected by a lower expression of the genes involved in disease and defence mechanisms. The results of this study provide a valuable set of data that may help to improve our understanding of the mechanisms employed by plants to tolerate toxic concentrations of metal in the soil.
Urinary felinine excretion in intact male cats is increased by dietary cystine
Hendriks, W.H. ; Rutherfurd-Markwick, K.J. ; Weidgraaf, K. ; Morton, R.H. ; Rogers, Q.R. - \ 2008
The British journal of nutrition 100 (2008)4. - ISSN 0007-1145 - p. 801 - 809.
glutathione s-transferases - felis-catus - cysteine dioxygenase - rats - inactivation - deficiency - methionine - precursor - glutamine - arginine
Felinine is a branched-chain sulfur amino acid present in the urine of certain Felidae, including domestic cats. The objective of the present study was to determine if additional cystine and/or dietary N would increase felinine and N-acetylfelinine excretion by intact male cats fed a low-protein (LP) diet. Feeding five adult intact male cats an LP diet (18·8% of metabolisable energy (ME) as protein) v. a high-protein diet (38·6% of ME as protein) resulted in a trend (P¼0·08) for decreased urinary felinine and no change in N-acetylfelinine excretion. In a 23 d study, when the LP diet was supplemented with L-cystine at 9·3 g/kg DM, urinary felinine:creatinine ratio showed a linear two-fold (121 %) increase (P,0·01) from 0·24 (SEM 0·05) to 0·53 (SEM 0·13) after 10 d. Subsequent feeding of the LP diet resulted in a decrease in felinine excretion to base levels. Plasma gglutamylfelinylglycine concentrations were consistent with the excretion of felinine. Supplementation of the LP diet with L-cystine (9·3 g/kg DM), dispensable amino acids and arginine to a second group (n 5) also resulted in a significant (P,0·01) but smaller (þ72 %) increase in the daily felinine:creatinine ratio (0·25 (SEM 0·04) to 0·43 (SEM 0·05)). The degree of felinine N-acetylation within groups was unaffected by dietary addition and withdrawal of amino acids. The results indicate that felinine synthesis is regulated by cystine availability, and that arginine may be physiologically important in decreasing felinine biosynthesis in intact male cats.
The FEMA GRAS assessment of a,b-unsaturated aldehydes and related substances used as flavor ingredients
Adams, T.B. ; Lucas-Gavin, C. ; Taylor, S.V. ; Waddell, W.J. ; Cohen, S.M. ; Feron, V.J. ; Goodman, J. ; Rietjens, I.M.C.M. ; Marnett, L.J. ; Portoghese, P.S. ; Smith, R.L. - \ 2008
Food and Chemical Toxicology 46 (2008)9. - ISSN 0278-6915 - p. 2935 - 2967.
sister-chromatid exchanges - glutathione s-transferases - acid nitrite invivo - long-term toxicity - sorbic acid - lipid-peroxidation - cytochrome-c - dna-damage - allyl alcohol - 1,n(2)-propanodeoxyguanosine adducts
This publication is the 12th in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Since then, the number of flavoring substances has grown to more than 2200 chemically-defined substances. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, toxicodynamics and toxicology. Scientific data relevant to the safety evaluation for the use of aliphatic, linear ¿,ß-unsaturated aldehydes and structurally related substances as flavoring ingredients are evaluated. The group of substances was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; their low level of flavor use; the rapid absorption and metabolism of low in vivo concentrations by well-recognized biochemical pathways; adequate metabolic detoxication at much higher levels of exposure in humans and animals; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies. While some of the compounds described here have exhibited positive in vitro genotoxicity results, evidence of in vivo genotoxicity and carcinogenicity occurs only under conditions in which animals are repeatedly and directly exposed to high irritating concentrations of the aldehyde. These conditions are not relevant to humans who consume ¿,ß-unsaturated aldehydes as flavor ingredients at low concentrations distributed in a food or beverage matrix.
The influence of fruit and vegetable consumption and genetic variation on NAD(P)H: quinone oxidoreductase (NQO1) phenotype in an endoscopy-based population
Tijhuis, M.J. ; Boerboom, A.M.J.F. ; Visker, M.H.P.W. ; Camp, E.B.G. op den; Nagengast, F.M. ; Tan, A.C.I.T.L. ; Rietjens, I.M.C.M. ; Kok, F.J. ; Aarts, J.M.M.J.G. ; Kampman, E. - \ 2008
Nutrition and Cancer 60 (2008)2. - ISSN 0163-5581 - p. 204 - 215.
glutathione s-transferases - dt-diaphorase activity - mitomycin-c treatment - colorectal-cancer - transcription factors - activator protein-1 - c609t polymorphism - brussels-sprouts - promoter region - cell-lines
NAD(P)H:quinone oxidoreductase (NQO1) is an inducible detoxification enzyme relevant for colorectal cancer biochemoprevention. We evaluated the influence of recent fruit and vegetable (F&V) consumption and polymorphisms in NQO1 and transcription factor NFE2L2 on rectal NQO1 phenotype and also whether white blood cell (WBC) NQO1 activity reflects rectal activity. Among 94 sigmoidoscopy patients, we assessed F&V consumption by dietary record and determined the NQO1 c.609C > T and g.-718A > G and NFE2L2 g.-650C > A, g.-684G > A, and g.-686A > G polymorphisms. NQO1 mRNA level was measured in rectal biopsies and NQO1 activity in rectal biopsies and WBC. Consumption of F&V did not yield higher mRNA level or activity but rather appeared to have a repressive effect. Rectal activity was higher among NQO1 609CC-genotypes as compared to 609CT-genotypes (P <0.0001; 609TT-genotypes were absent), whereas mRNA was higher among 609CT-genotypes (P <0.001). mRNA and activity correlated among NQO1 609CC-genotypes (r = .50, P = 0.0001) but not among 609CT-genotypes (r = .14, P = 0.45). The NFE2L2-684A-allele was associated with higher mRNA levels (P = <0.05). The other polymorphisms did not affect phenotype significantly. WBC and rectal activity did not correlate. In conclusion, genetic variation, especially the NQO1 609C > T polymorphism, is a more important predictor of rectal NQO1 phenotype than F&V consumption. WBC NQO1 activity is not a good surrogate for rectal activity.
GSTP1 and GSTA1 Polymorphisms interact with Cruciferous Vegetable Intake in Colorectal Adenoma Risk
Tijhuis, M.J. ; Wark, P.A. ; Aarts, J.M.M.J.G. ; Visker, M.H.P.W. ; Nagengast, F.M. ; Kok, F.J. ; Kampman, E. - \ 2005
Cancer Epidemiology Biomarkers & Prevention 14 (2005)12. - ISSN 1055-9965 - p. 2943 - 2951.
glutathione s-transferases - brassica vegetables - colon-cancer - dietary isothiocyanates - genetic polymorphisms - human-lymphocytes - prostate-cancer - rectal cancers - consumption - fruit
The possible interplay between cruciferous vegetable consumption, functional genetic variations in glutathione S-transferases (GST) M1, T1, P1, and A1, and colorectal adenomas, was investigated in a Dutch case-control study. The GSTM1 and GSTT1 deletion polymorphisms, and the single nucleotide polymorphisms in GSTP1 (A313G) and in GSTA1 (C-69T) were assessed among 746 cases who developed colorectal adenomas and 698 endoscopy-based controls without any type of colorectal polyps. High and low cruciferous vegetable consumption was defined based on a median split in the control group. High consumption was slightly positively associated with colorectal adenomas [odds ratio (OR) 1.15; 95% confidence interval, 0.92-1.44]. For GSTP1, a positive association with higher cruciferous vegetable intake was only apparent in individuals with the low-activity GSTP1 genotype (GG genotype, OR 1.94; 95% confidence interval, 1.02-3.69). This interaction was more pronounced in men, with higher age and with higher meat intake. The GSTA1 polymorphism may have a modifying role as well: the OR for higher intake compared with lower intake was 1.57 (0.93-2.65) for individuals homozygous for the low expression variant (TT genotype). This seemed to be stronger with younger age and higher red meat intake. Cruciferous vegetable consumption and the combined GSTA1 and GSTP1 genotypes showed a statistically significant interaction (P = 0.034). The GSTM1 and GSTT1 genotypes did not seem to modify the association between cruciferous vegetable intake and colorectal adenomas. In conclusion, GSTP1 and GSTA1 genotypes might modulate the association between cruciferous vegetable intake and colorectal adenomas. (Cancer Epidemiol Biomarkers Prev 2005;14(12):2943¿51)
Proteome analysis reveals novel proteins associated with proliferation and differentiation of the colorectal cancer cell line Caco-2
Stierum, R. ; Gaspari, M. ; Dommels, Y.E.M. ; Ouatas, T. ; Pluk, H. ; Jespersen, S. ; Vogels, J. ; Verhoeckx, K. ; Groten, J. ; Ommen, B. van - \ 2003
Biochimica et Biophysica Acta. Proteins & Proteomics 1650 (2003)1-2. - ISSN 1570-9639 - p. 73 - 91.
creatine-kinase bb - glutathione s-transferases - alzheimers-disease brain - nitric-oxide synthase - acid-binding-protein - human colon-cancer - down-regulation - 1,25-dihydroxyvitamin d-3 - gene-expression - actin dynamics
Here, we describe a proteomics approach to study protein expression changes in differentiating Caco-2 cells. Caco-2 is a colorectal carcinoma cell line, which upon differentiation loses its tumorigenic phenotype and displays characteristics of mature enterocytes, including brush borders with microvilli. Cells were grown in culture flasks and harvested at different stages of differentiation (days post-confluence: ¿3, 0, 3, 7, 10, 14, and 18). Two-dimensional gel electrophoresis was used to analyse proteome changes. Approximately 1400 protein spots were detected within the Caco-2 proteome, within the pH 4¿7 range. Two-dimensional gel electrophoresis allowed for the detection of 18 proteins from which the levels of expression were found to be associated with differentiation. Of these proteins, 11 were identified by means of MALDI-TOF or NANO-ESI-MS/MS mass spectrometry and include liver fatty acid binding protein (FABL), three forms of ¿-enolase (ENOA), nucleoside diphosphate kinase A (NDKA), cofilin-1 (COF1), translationally controlled tumour protein (TCTP), mitochondrial 60-kDa heat shock protein (CH60), probable protein disulfide isomerase (ER60), creatine kinase B (KCRB), and glutathione S-transferase ¿ (GTA1). Thus, proteomics revealed that the differentiation-related change in phenotype of Caco-2 involves changes in a variety of distinct biochemical pathways. Some of these proteins have not been shown before to be associated with Caco-2 differentiation (ER60; COF1; CH60; NDKA; TCTP and ENOA). Therefore, processes related to protein folding and disulfide bridge formation, cytoskeleton formation and maintenance, nucleotide metabolism, glycolysis as well as tumorigenesis-associated proteins may be involved in Caco-2 differentiation. Changes in the expression of CH60, TCTP, GTA1, NDKA, and FABL have also been reported to be associated with in vivo colon carcinogenesis. These findings illustrate that a combination of proteomics and cell culture is a useful approach to find markers for Caco-2 differentiation, which could contribute to the comprehension of the process of colon carcinogenesis.
Plant Foods versus Compounds in Carcinogenesis: Observational versus Experimental Human Studies
Kampman, E. ; Arts, I.C.W. ; Hollman, P.C.H. - \ 2003
International Journal for Vitamin and Nutrition Research 73 (2003)2. - ISSN 0300-9831 - p. 70 - 78.
glutathione s-transferases - iowa womens health - colorectal-cancer - serum enterolactone - brussels-sprouts - lung-cancer - dietary differences - flavonoid intake - phyto-estrogens - breast-cancer
The protective role of plant foods and its constituents in cancer prevention is under renewed debate since the results of recent observational studies on colorectal cancer as well as large-scale human experimental studies on colorectal adenoma recurrence are disappointing. However, most short-term experimental human studies do show that plant foods favourably modulate potential cancer-preventive mechanisms. Which methodological pitfalls may explain the inconsistencies within and between different study designs? What are the advantages and limitations of the different study approaches? Observational studies do have the advantage to study the population at large with ultimate disease as the study endpoint. These studies are limited by the difficulty to estimate intake of individual compounds by questionnaires and the lack of biological markers of relevant exposure. Controlled experimental short-term studies in humans rely on biological markers of disease as intermediate endpoints. Relatively low sensitivity and specificity of these markers may complicate extrapolation of results. In the case of long-term and large-scale human intervention studies with disease endpoints, issues such as time, dose and duration of intervention, compliance and choice of the study population influence the interpretation of results. An integrated approach combining designs, and implementing new techniques to identify biomarkers, may clarify the role of plant foods in carcinogenesis.
Quenching of quercetin quinone/quinone methides by different thiolate scavengers: stability and reversibility of conjugate formation
Awad, H.M. ; Boersma, M.G. ; Boeren, J.A. ; Bladeren, P.J. van; Vervoort, J.J.M. ; Rietjens, I.M.C.M. - \ 2003
Chemical Research in Toxicology 16 (2003)7. - ISSN 0893-228X - p. 822 - 831.
glutathione s-transferases - irreversible inhibition - lipid-peroxidation - mutagenic activity - quinone methide - active-site - o-quinones - oxidation - flavonoids - antioxidant
Oxidation of flavonoids with a catechol structural motif in their B ring leads to formation of flavonoid quinone/quinone methides, which rapidly react with GSH to give reversible glutathionyl flavonoid adducts. Results of the present study demonstrate that as a thiol-scavenging agent for this reaction Cys is preferred over GSH and N-acetyleysteine. The preferential scavenging by Cys over GSH reported in the present study appeared not to provide a basis for detection of thiol-based flavonoid conjugates in biological systems. This is because physiological concentrations of GSH are substantially higher than those of Cys, which was shown to shift the balance of thiol conjugate formation in favor of glutathionyl adduct formation. Furthermore, the cysteinyl quercetin adducts, although not showing the reversible nature of the glutathionyl conjugates, appeared nevertheless to be unstable. Thus, as a biomarker for formation of reactive quercetin quinone/quinone methides in biological systems, detection of the glutathionyl conjugates or the N-acetyleysteinyl conjugates derived from them should still be the method of choice. At GSH levels that dominate the level of other cellular thiol groups, covalent addition of the quinone to other cellular thiol groups may be efficiently prevented. However, various tissues are known to contain higher levels of protein-bound sulfhydryl moieties than of nonprotein sulfhydryl groups, the latter consisting of especially GSH. Thus, the results of the present study indicate that in biological systems covalent addition of quercetin quinone methide to tissue protein sulfhydryl groups can be expected. The transient nature of these adducts, as shown for all three types of thiol quercetin adducts in the present study, will, however, also result in a transient nature of the protein-bound quercetin adducts to be expected. Because stability of the various thiol quercetin adducts appeared a matter of minutes to hours instead of days, this rapid transient nature of possible quercetin quinone methide adducts may also restrict the ultimate toxicity to be expected from the quercetin quinone/quinone methides.