RESUMEN
Herein for the first time a novel acid phosphatase from the seedlings of Cichorium intybus was purified to homogeneity by using various chromatographic techniques (salt precipitation, ion exchange, size exclusion and affinity chromatography) and thermodynamically characterized. The molecular mass of purified enzyme (66 kDa) was determined by SDS-PAGE under denaturing and non-denaturing conditions and by gel-filtration confirmed as dimer of molecular mass 130 kDa. The Michaelis-Menten (Km) constant for -p-NPP (0.3 mM) and (7.6 µmol/min/mg) Vmax. The enzyme was competitively inhibited by phosphate, molybdate and vanadate. Phenyl phosphate, É and ß-glycero-phosphate and-p-NPP were found to be good substrate. When temperature increased from (55 °C to 75 °C), the deactivation rate constant (kd) was increased (0.1 to 4.6 min-1) and half- life was decreased from 630 min to 15 min. Various thermal denaturation parameters; change in enthalpy (ΔH°), change in entropy (ΔS°) and change in free energy (ΔG°) were found 121.93 KJ·mol-1, 72.45 KJ·mol-1 and 98.08 KJ·mol-1 respectively, confirming that acid phosphatase undergoes a significant process of unfolding during deactivation. The biochemical properties of acid phosphatase from C. intybus on the behalf of biological activity and its relationship to pH variations, thermal deactivation and kinetics parameters provide an insight into its novel features.
Asunto(s)
Fosfatasa Ácida/química , Fosfatasa Ácida/aislamiento & purificación , Cichorium intybus/química , Cichorium intybus/enzimología , Cichorium intybus/metabolismo , Cromatografía en Gel/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Fosfatos , Plantones/química , Temperatura , TermodinámicaRESUMEN
The thermal inactivation kinetics of enzymes, including polyphenol oxidase (PPO) and peroxidase (POD), in chicory (Cichorium intybus L.) leaves were evaluated. In addition, the influences of different drying techniques (shade drying, hot air drying and freeze drying) on the phenolic profiles and antioxidant activities of chicory leaves were determined. The antioxidant activities of chicory leaves were evaluated on the basis of their 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing power, and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity. The results showed that the activation energy for PPO and POD inactivation were 123.00 kJ/mol and 78.99 kJ/mol, respectively. Preliminary treatment with hot water for 3 min at 90 °C was beneficial for preserving the phenolics present in fresh leaves. Hot air drying was better for the phenolics preservation. The hot air-dried and freeze-dried leaves possessed good antioxidant activities. The leaves with higher phenolics contents had better antioxidant activities, which indicated that the preservation of the phenolics was important for maintaining the antioxidant activity of chicory leaves.
Asunto(s)
Antioxidantes/metabolismo , Cichorium intybus/metabolismo , Liofilización/métodos , Calor , Fenoles/metabolismo , Hojas de la Planta/metabolismo , Benzotiazoles/metabolismo , Compuestos de Bifenilo/metabolismo , Catecol Oxidasa/metabolismo , Cichorium intybus/enzimología , Activación Enzimática , Cinética , Peroxidasas/metabolismo , Picratos/metabolismo , Hojas de la Planta/enzimología , Ácidos Sulfónicos/metabolismoRESUMEN
Cichorium intybus (common chicory), a perennial plant, common in anthropogenic sites, has been the object of a multitude of studies in recent years due to its high content of antioxidants utilized in pharmacy and food industry. Here, the role of arbuscular mycorrhizal fungi (AMF) in the biosynthesis of plant secondary metabolites and the activity of enzymatic antioxidants under toxic metal stress was studied. Plants inoculated with Rhizophagus irregularis and non-inoculated were grown on non-polluted and toxic metal enriched substrata. The results presented here indicate that AMF improves chicory fitness. Fresh and dry weight was found to be severely affected by the fungi and heavy metals. The concentration of hydroxycinnamates was increased in the shoots of mycorrhizal plants cultivated on non-polluted substrata, but no differences were found in plants cultivated on metal enriched substrata. The activity of SOD and H2O2 removing enzymes CAT and POX was elevated in the shoots of mycorrhizal plants regardless of the cultivation environment. Photochemical efficiency of inoculated chicory was significantly improved. Our results indicate that R. irregularis inoculation had a beneficial role in sustaining the plants ability to cope with the deleterious effects of metal toxicity.
Asunto(s)
Antioxidantes/metabolismo , Cichorium intybus/efectos de los fármacos , Cichorium intybus/metabolismo , Glomeromycota/fisiología , Metales/toxicidad , Micorrizas/fisiología , Fitoquímicos/biosíntesis , Cichorium intybus/enzimología , Cichorium intybus/microbiología , Contaminantes Ambientales/toxicidad , Peróxido de Hidrógeno/metabolismoRESUMEN
KEY MESSAGE: Nucleotidic polymorphisms were identified in fructan exohydrolases genes which are statistically associated with enhanced susceptibility to post-harvest inulin depolymerization. Industrial chicory (Cichorium intybus L.) root is the main commercial source of inulin, a linear fructose polymer used as dietary fiber. Post-harvest, inulin is depolymerized into fructose which drastically increases processing cost. To identify genetic variations associated with enhanced susceptibility to post-harvest inulin depolymerization and related free sugars content increase, we used a candidate-gene approach focused on inulin and sucrose synthesis and degradation genes, all members of the family 32 of glycoside hydrolases (GH32). Polymorphism in these genes was first investigated by carrying out EcoTILLING on two groups of chicory breeding lines exhibiting contrasted response to post-harvest inulin depolymerization. This allowed the identification of polymorphisms significantly associated with depolymerization in three fructan exohydrolase genes (FEH). This association was confirmed on a wider panel of 116 unrelated families in which the FEH polymorphism explained 35 % of the post-harvest variance for inulin content, 36 % of variance for sucrose content, 18 % for inulin degree of polymerization, 23 % for free fructose content and 22 % for free glucose content. These polymorphisms were associated with significant post-harvest changes of inulin content, inulin chain length and free sugars content.
Asunto(s)
Cichorium intybus/genética , Genes de Plantas , Glicósido Hidrolasas/genética , Inulina/metabolismo , Polimorfismo Genético , Cichorium intybus/enzimología , Estudios de Asociación Genética , PolimerizacionRESUMEN
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.
Asunto(s)
Vías Biosintéticas , Nicotiana/metabolismo , Sesquiterpenos/metabolismo , Levaduras/metabolismo , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Cichorium intybus/enzimología , Cichorium intybus/genética , Cromatografía Liquida/métodos , Cisteína/química , Cisteína/metabolismo , Sistema Enzimático del Citocromo P-450/clasificación , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Glutatión/química , Glutatión/metabolismo , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Estructura Molecular , Oxidación-Reducción , Oxidorreductasas/clasificación , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sesquiterpenos/química , Sesquiterpenos de Germacrano/química , Sesquiterpenos de Germacrano/metabolismo , Tanacetum parthenium/enzimología , Tanacetum parthenium/genética , Nicotiana/genética , Transformación Genética , Levaduras/genéticaRESUMEN
Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene.
Asunto(s)
Cichorium intybus/enzimología , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas de Plantas/metabolismo , Sesquiterpenos/metabolismo , Biocatálisis , Cichorium intybus/genética , Clonación Molecular , Sistema Enzimático del Citocromo P-450/genética , ADN Complementario/química , ADN Complementario/genética , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Datos de Secuencia Molecular , Oxidación-Reducción , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Proteínas de Plantas/genética , Sesquiterpenos Policíclicos , Análisis de Secuencia de ADN , Sesquiterpenos/química , Sesquiterpenos de Germacrano/metabolismo , Estereoisomerismo , Levaduras/genética , Levaduras/metabolismoRESUMEN
Fifteen per cent of higher plants accumulate fructans. Plant development, nutritional status and stress exposure all affect fructan metabolism, and while fructan biochemistry is well understood, knowledge of its regulation has remained fragmentary. Here, we have explored chicory (Cichorium intybus) hairy root cultures (HRCs) to study the regulation of fructan metabolism in sink tissues in response to environmental cues. In standard medium (SM), HRCs did not accumulate inulin. However, upon transfer to high-carbon (C)/low-nitrogen (N) medium, expression of sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT) was strongly induced and inulin accumulated. Upon return to SM, inulin was degraded, together with a coordinate decline of 1-SST and 1-FFT expression. In HRCs, cold-induced expression of fructan 1-exohydrolases (1-FEH I and IIa) was similar to cold induction in taproots, even in the absence of accumulated inulin. For high-C/low-N induction of 1-SST and 1-FFT, and cold induction of 1-FEH I and IIa, the signaling pathways were addressed. While 1-SST and 1-FFT induction was similarly prevented by inhibitors of Ca(2+) signaling, protein kinases and phosphatases, cold induction of 1-FEH I and IIa revealed distinct signaling pathways. In summary, this study has established chicory HRCs as a convenient experimental system with which to study the regulation of fructan active enzyme (FAZY) expression in heterotrophic cells.
Asunto(s)
Cichorium intybus/metabolismo , Fructanos/metabolismo , Raíces de Plantas/metabolismo , Transporte Biológico/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Cichorium intybus/efectos de los fármacos , Cichorium intybus/enzimología , Cichorium intybus/genética , Frío , Medios de Cultivo , Regulación hacia Abajo/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Hexosiltransferasas/biosíntesis , Hexosiltransferasas/genética , Inulina/metabolismo , Nitrógeno/farmacología , Fosfoproteínas Fosfatasas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Proteínas Quinasas/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Plant fructan active enzymes (FAZYs), including the enzymes involved in inulin metabolism, namely sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100) and fructan 1-exohydrolase (1-FEH; EC 3.2.1.153), are evolutionarily related to acid invertases (AIs), that is, plant cell wall invertase (CWI) and vacuolar invertase (VI). Acid invertases are post-translationally controlled by proteinaceous inhibitors. Whether FAZYs are subject to similar controls is not known. To probe their possible interactions with invertase inhibitors, we transiently expressed chicory (Cichorium intybus) FAZYs, as well as several previously characterized invertase inhibitors from nonfructan species, and the C. intybus cell wall/vacuolar inhibitor of fructosidase (CiC/VIF), a putative invertase inhibitor of a fructan-accumulating plant, in leaves of Nicotiana benthamiana. Leaf extracts containing recombinant, enzymatically active FAZYs were used to explore the interaction with invertase inhibitors. Neither heterologous inhibitors nor CiC/VIF affected FAZY activities. CiC/VIF was confirmed as an AI inhibitor with a stronger effect on CWI than on VI. Its expression in planta was developmentally regulated (high in taproots, and undetectable in leaves and flowers). In agreement with its target specificities, CiC/VIF was associated with the cell wall. It is concluded that subtle structural differences between AIs and FAZYs result in pronounced selectivity of inhibitor action.
Asunto(s)
Cichorium intybus/enzimología , Inhibidores Enzimáticos/metabolismo , Fructanos/metabolismo , Homología Estructural de Proteína , beta-Fructofuranosidasa/antagonistas & inhibidores , Secuencia de Aminoácidos , Cichorium intybus/genética , Clonación Molecular , Inhibidores Enzimáticos/química , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes/metabolismo , Especificidad de la Especie , Nicotiana/enzimología , Nicotiana/genética , beta-Fructofuranosidasa/genéticaRESUMEN
The hydrolytic plant enzymes of family 32 of glycoside hydrolases (GH32), including acid cell wall type invertases (EC 3.2.1.26), fructan 1-exohydrolases (1-FEH; EC 3.2.1.153) and fructan 6-exohydrolases (6-FEH; EC 3.2.1.154), are very similar at the molecular and structural levels, but are clearly functionally different. The work presented here aims at understanding the evolution of enzyme specificity and functional diversity in this family by means of site-directed mutagenesis. It is demonstrated for the first time that invertase activity can be introduced in an S101L mutant of chicory (Cichorium intybus) 1-FEH IIa by influencing the orientation of Trp 82. At high sucrose and enzyme concentrations, a shift is proposed from a stable inhibitor configuration to an unstable substrate configuration. In the same way, invertase activity was introduced in Beta vulgaris 6-FEH by introducing an acidic amino acid in the vicinity of the acid-base catalyst (F233D mutant), creating a beta-fructofuranosidase type of enzyme with dual activity against sucrose and levan. As single amino acid substitutions can influence the donor substrate specificity of FEHs, it is predicted that plant invertases and FEHs may have diversified by introduction of a very limited number of mutations in the common ancestor.
Asunto(s)
Beta vulgaris/enzimología , Cichorium intybus/enzimología , Glicósido Hidrolasas/genética , Sacarosa/química , Sacarosa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Ingeniería Genética , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Unión Proteica , Conformación Proteica , Especificidad por SustratoRESUMEN
Recently, the three-dimensional structure of chicory (Cichorium intybus) fructan 1-exohydrolase (1-FEH IIa) in complex with its preferential substrate, 1-kestose, was determined. Unfortunately, no such data could be generated with high degree of polymerization (DP) inulin, despite several soaking and cocrystallization attempts. Here, site-directed mutagenesis data are presented, supporting the presence of an inulin-binding cleft between the N- and C-terminal domains of 1-FEH IIa. In general, enzymes that are unable to degrade high DP inulins contain an N-glycosylation site probably blocking the cleft. By contrast, inulin-degrading enzymes have an open cleft configuration. An 1-FEH IIa P294N mutant, introducing an N-glycosylation site near the cleft, showed highly decreased activity against higher DP inulin. The introduction of a glycosyl chain most probably blocks the cleft and prevents inulin binding and degradation. Besides cell wall invertases, fructan 6-exohydrolases (6-FEHs) also contain a glycosyl chain most probably blocking the cleft. Removal of this glycosyl chain by site-directed mutagenesis in Arabidopsis thaliana cell wall invertase 1 and Beta vulgaris 6-FEH resulted in a strong decrease of enzymatic activities of the mutant proteins. By analogy, glycosylation of 1-FEH IIa affected overall enzyme activity. These data strongly suggest that the presence or absence of a glycosyl chain in the cleft is important for the enzyme's stability and optimal conformation.
Asunto(s)
Cichorium intybus/enzimología , Glicósido Hidrolasas/metabolismo , Inulina/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/enzimología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Beta vulgaris/enzimología , Sitios de Unión , Pared Celular/enzimología , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Glicosilación , Inulina/química , Inulina/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estructura Terciaria de Proteína , Alineación de Secuencia , Especificidad por Sustrato , beta-Fructofuranosidasa/química , beta-Fructofuranosidasa/genética , beta-Fructofuranosidasa/metabolismoRESUMEN
* Invertases and fructan exohydrolases (FEHs) fulfil important physiological functions in plants. Sucrose is the typical substrate for invertases and bacterial levansucrases but not for plant FEHs, which are usually inhibited by sucrose. * Here we report on complexes between chicory (Cichorium intybus) 1-FEH IIa with the substrate 1-kestose and the inhibitors sucrose, fructose and 2,5 dideoxy-2,5-imino-D-mannitol. Comparisons with other family GH32 and 68 enzyme-substrate complexes revealed that sucrose can bind as a substrate (invertase/levansucrase) or as an inhibitor (1-FEH IIa). * Sucrose acts as inhibitor because the O2 of the glucose moiety forms an H-linkage with the acid-base catalyst E201, inhibiting catalysis. By contrast, the homologous O3 of the internal fructose in the substrate 1-kestose forms an intramolecular H-linkage and does not interfere with the catalytic process. Mutagenesis showed that W82 and S101 are important for binding sucrose as inhibitor. * The physiological implications of the essential differences in the active sites of FEHs and invertases/levansucrases are discussed. Sucrose-inhibited FEHs show a K(i) (inhibition constant) well below physiological sucrose concentrations and could be rapidly activated under carbon deprivation.
Asunto(s)
Cichorium intybus/enzimología , Glicósido Hidrolasas/química , Proteínas de Plantas/química , Trisacáridos/química , Sitios de Unión , Secuencia de Carbohidratos , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Glicósido Hidrolasas/antagonistas & inhibidores , Glicósido Hidrolasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/genética , Conformación Proteica , Sacarosa/farmacología , Trisacáridos/metabolismoRESUMEN
The major component of chicory (Chicorium intybus) root is inulin, which is a polymer of fructose. Inulin production from chicory is hampered by the enzyme fructan 1-exohydrolase (1-FEH) that degrades inulin and limits its yield. Increased FEH activity results in massive breakdown of fructan and production of Fructose and inulo-n-oses. The latter phenomena are to be avoided for industrial fructan production. RNA silencing, which is termed post-transcriptional gene silencing (PTGS) in plants, is an RNA degradation process through sequence specific nucleotide interactions induced by double-stranded RNA. For genetic improvement of crop plants, RNAi has advantages over antisense-mediated gene silencing and co-suppression, in terms of its efficiency and stability. We are generating a transgenic chicory plants with suppressed FEH (exohydrolas) genes using RNAi resulting in supressed inulin degradation. A small but important part of the construct is a sequence unique for the target gene (exons) or genes,which were cloned. The hairpin constructs were made and chicory was transformed by Agrobacterium tumifaciense, strain (C58C1). The transgenics should be select and check by means of molecular techniques.
Asunto(s)
Cichorium intybus/enzimología , Cichorium intybus/genética , Glicósido Hidrolasas/metabolismo , Insulina/metabolismo , Interferencia de ARN , Metabolismo de los Hidratos de Carbono , Cichorium intybus/crecimiento & desarrollo , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Valor Nutritivo , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrolloRESUMEN
Thermotoga maritima invertase (beta-fructosidase), a member of the glycoside hydrolase family GH-32, readily releases beta-D-fructose from sucrose, raffinose and fructan polymers such as inulin. These carbohydrates represent major carbon and energy sources for prokaryotes and eukaryotes. The invertase cleaves beta-fructopyranosidic linkages by a double-displacement mechanism, which involves a nucleophilic aspartate and a catalytic glutamic acid acting as a general acid/base. The three-dimensional structure of invertase shows a bimodular enzyme with a five bladed beta-propeller catalytic domain linked to a beta-sandwich of unknown function. In the present study we report the crystal structure of the inactivated invertase in interaction with the natural substrate molecule alpha-D-galactopyranosyl-(1,6)-alpha-D-glucopyranosyl-beta-D-fructofuranoside (raffinose) at 1.87 A (1 A=0.1 nm) resolution. The structural analysis of the complex reveals the presence of three binding-subsites, which explains why T. maritima invertase exhibits a higher affinity for raffinose than sucrose, but a lower catalytic efficiency with raffinose as substrate than with sucrose.
Asunto(s)
Rafinosa/química , Rafinosa/metabolismo , Thermotoga maritima/enzimología , beta-Fructofuranosidasa/química , beta-Fructofuranosidasa/metabolismo , Aspergillus/enzimología , Sitios de Unión , Conformación de Carbohidratos , Dominio Catalítico , Cichorium intybus/enzimología , Cristalografía por Rayos X , Activación Enzimática , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Enlace de Hidrógeno , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Homología Estructural de Proteína , Especificidad por Sustrato , Thermotoga maritima/genética , beta-Fructofuranosidasa/genéticaRESUMEN
Pectins are major components of the primary plant cell wall. They can be both methylesterified and acetylesterified and de-esterification occurs by specific esterases. Proteins extracted by NaCl treatment from root cell walls of two chicory varieties (Cichorium intybus L. cv. Nausica and Arancha) sampled in an experimental field every 2 weeks between July 2002 and January 2003 were analysed by isoelectrofocalization, semi-denaturing SDS-PAGE, and quantitative assays for their esterase activity. Zymograms showed that chicory root pectin methylesterases belong to a multigene family. The isoelectric points of the pectin methylesterase isoforms ranged from pI 3.8 to pI 9.0. Concerning acetylesterases, only acidic isoforms between pI 4.1 and pI 5.2 were observed, but a large polymorphism of this class of enzymes could be identified in one variety. The results indicate that the root pectin methylesterase activity of the Nausica variety was correlated with ambient temperature, while no significant effect of temperature could be detected on any acetylesterase isoform.
Asunto(s)
Acetilesterasa/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Pared Celular/enzimología , Cichorium intybus/enzimología , Raíces de Plantas/enzimología , Electroforesis en Gel de Poliacrilamida , Focalización Isoeléctrica , Isoenzimas/metabolismo , Análisis de Regresión , Estaciones del AñoRESUMEN
A water-soluble lipoxygenase enzyme (EC 1.13.11.12; LOX) occurring in the red cultivar produced in the geographical area of Chioggia (Italy) of Cichorium intybus var. silvestre was isolated and characterized. The molecular mass of the enzyme was estimated to be 74,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. The isoelectric point was pH 6.85. The optimum values of pH, ionic strength, and temperature, shown by isoresponse surface calculated by a randomized multilevel factorial design, were 7.58, 30 mM, and 38.5 degrees C, respectively. The enzyme showed high specificity toward linoleic acid, and the study of the variation of linoleic acid concentration between 30 and 300 microM, in the presence of Tween 20 at a concentration lower than the critical micelle concentration (0.01 v/v), resulted in a typical Michaelis-Mentem curve with KM and Vmax values of 1.49 x 10(-4) M and 2.049 microM min(-1) mg(-1), respectively. The biochemical properties, the kinetic parameters found, and the carotene-bleaching activity shown in aerobic conditions seem to indicate that the isolated enzyme is a lipoxygenase type III according to the indications given for soybean isoenzymes.
Asunto(s)
Cichorium intybus/enzimología , Lipooxigenasa/aislamiento & purificación , Lipooxigenasa/metabolismo , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Lipooxigenasa/química , Peso Molecular , Concentración Osmolar , TemperaturaRESUMEN
Fructan 1-exohydrolase, an enzyme involved in fructan degradation, belongs to the glycosyl hydrolase family 32. The structure of isoenzyme 1-FEH IIa from Cichorium intybus is described at a resolution of 2.35 A. The structure consists of an N-terminal fivefold beta-propeller domain connected to two C-terminal beta-sheets. The putative active site is located entirely in the beta-propeller domain and is formed by amino acids which are highly conserved within glycosyl hydrolase family 32. The fructan-binding site is thought to be in the cleft formed between the two domains. The 1-FEH IIa structure is compared with the structures of two homologous but functionally different enzymes: a levansucrase from Bacillus subtilis (glycosyl hydrolase family 68) and an invertase from Thermotoga maritima (glycosyl hydrolase family 32).
Asunto(s)
Cichorium intybus/enzimología , Glicósido Hidrolasas/química , Secuencia de Aminoácidos , Bacillus subtilis/enzimología , Sitios de Unión , Secuencia Conservada , Hexosiltransferasas/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Thermotoga maritima/enzimología , Difracción de Rayos X , beta-Fructofuranosidasa/químicaRESUMEN
The gene for a recently identified cDNA, 1-FEH IIa, encoding a fructan 1-exohydrolase was isolated and cloned from Cichorium intybus and a 1149 bp promoter fragment was characterized. An analysis of the genomic 1-FEH IIa sequence indicated that the gene (FEHIIa) consists of six introns and seven exons, which is similar to plant invertase genes. Like invertase genes, FEHIIa also contains the 9 nt mini-exon encoding the tripeptide DPN. A database search for cis-acting response elements within its promoter identified multiple elements that appear to have relevance to cold-induced expression of the gene in field-grown roots. Promoter analysis by transient expression assay demonstrated that the FEHIIa gene promoter is highly expressed in etiolated Cichorium leaves and cold-stored roots, which correlated well with the high level expression detected by RNA blot analysis. Cold also enhanced FEHIIa reporter gene expression in green leaves, however, the reporter gene activity was much lower compared with similar induction experiments in etiolated leaves. Promoter deletion analysis demonstrated the presence of potential cold-responsive ABRE and/or CRT/DRE elements in the -22 to -172 region, while regions -933 to -717 and -493 to -278 contain elements that can down-regulate expression at the conditions used. Characterization of the FEHIIa promoter may provide tools to study cold-induced expression and to increase freezing tolerance in agricultural crops.
Asunto(s)
Cichorium intybus/enzimología , Glicósido Hidrolasas/genética , Secuencia de Bases , Cichorium intybus/genética , Cichorium intybus/fisiología , Clonación Molecular , Frío , ADN de Plantas/química , ADN de Plantas/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glicósido Hidrolasas/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Raíces de Plantas/fisiología , Análisis de Secuencia de ADN , Eliminación de Secuencia/genética , Eliminación de Secuencia/fisiología , Especificidad por SustratoRESUMEN
Fructan 1-exohydrolase IIa (1-FEH IIa), a plant enzyme involved in fructan breakdown, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. The crystals are tetragonal, belonging to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 139.83, b = 139.83, c = 181.94 A. Calculation of the Matthews coefficient indicates there to be two or three molecules in the asymmetric unit. Synchrotron radiation was used to collect a complete native data set to a resolution of 2.35 A.
Asunto(s)
Cichorium intybus/enzimología , Glicósido Hidrolasas/química , Proteínas de Plantas/química , Cristalización , Cristalografía por Rayos X , Fructanos/metabolismo , Glicósido Hidrolasas/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
Remarkably, within the Asteraceae, a species-specific fructan pattern can be observed. Some species such as artichoke (Cynara scolymus) and globe thistle (Echinops ritro) store fructans with a considerably higher degree of polymerization than the one observed in chicory (Cichorium intybus) and Jerusalem artichoke (Helianthus tuberosus). Fructan:fructan 1-fructosyltransferase (1-FFT) is the enzyme responsible for chain elongation of inulin-type fructans. 1-FFTs were purified from chicory and globe thistle. A comparison revealed that chicory 1-FFT has a high affinity for sucrose (Suc), fructose (Fru), and 1-kestose as acceptor substrate. This makes redistribution of Fru moieties from large to small fructans very likely during the period of active fructan synthesis in the root when import and concentration of Suc can be expected to be high. In globe thistle, this problem is avoided by the very low affinity of 1-FFT for Suc, Fru, and 1-kestose and the higher affinity for inulin as acceptor substrate. Therefore, the 1-kestose formed by Suc:Suc 1-fructosyltransferase is preferentially used for elongation of inulin molecules, explaining why inulins with a much higher degree of polymerization accumulate in roots of globe thistle. Inulin patterns obtained in vitro from 1-kestose and the purified 1-FFTs from both species closely resemble the in vivo inulin patterns. Therefore, we conclude that the species-specific fructan pattern within the Asteraceae can be explained by the different characteristics of their respective 1-FFTs. Although 1-FFT and bacterial levansucrases clearly differ in their ability to use Suc as a donor substrate, a kinetic analysis suggests that 1-FFT also works via a ping-pong mechanism.
Asunto(s)
Cichorium intybus/enzimología , Echinops (Planta)/enzimología , Hexosiltransferasas/metabolismo , Inulina/biosíntesis , Proteínas de Plantas , Asteraceae/enzimología , Cichorium intybus/metabolismo , Echinops (Planta)/metabolismo , Fructanos/biosíntesis , Fructosa/metabolismo , Hexosiltransferasas/aislamiento & purificación , Cinética , Modelos Biológicos , Especificidad por Sustrato , Sacarosa/metabolismo , Trisacáridos/metabolismoRESUMEN
Chicory (Cichorium intybus) sesquiterpene lactones were recently shown to be derived from a common sesquiterpene intermediate, (+)-germacrene A. Germacrene A is of interest because of its key role in sesquiterpene lactone biosynthesis and because it is an enzyme-bound intermediate in the biosynthesis of a number of phytoalexins. Using polymerase chain reaction with degenerate primers, we have isolated two sesquiterpene synthases from chicory that exhibited 72% amino acid identity. Heterologous expression of the genes in Escherichia coli has shown that they both catalyze exclusively the formation of (+)-germacrene A, making this the first report, to our knowledge, on the isolation of (+)-germacrene A synthase (GAS)-encoding genes. Northern analysis demonstrated that both genes were expressed in all chicory tissues tested albeit at varying levels. Protein isolation and partial purification from chicory heads demonstrated the presence of two GAS proteins. On MonoQ, these proteins co-eluted with the two heterologously produced proteins. The K(m) value, pH optimum, and MonoQ elution volume of one of the proteins produced in E. coli were similar to the values reported for the GAS protein that was recently purified from chicory roots. Finally, the two deduced amino acid sequences were modeled, and the resulting protein models were compared with the crystal structure of tobacco (Nicotiana tabacum) 5-epi-aristolochene synthase, which forms germacrene A as an enzyme-bound intermediate en route to 5-epi-aristolochene. The possible involvement of a number of amino acids in sesquiterpene synthase product specificity is discussed.