RESUMO
Lycibarbarspermidines are unusual phenolamide glycosides characterized by a dicaffeoylspermidine core with multiple glycosyl substitutions, and serve as a major class of bioactive ingredients in the wolfberry. So far, little is known about the enzymatic basis of the glycosylation of phenolamides including dicaffeoylspermidine. Here, we identify five lycibarbarspermidine glycosyltransferases, LbUGT1-5, which are the first phenolamide-type glycosyltransferases and catalyze regioselective glycosylation of dicaffeoylspermidines to form structurally diverse lycibarbarspermidines in wolfberry. Notably, LbUGT3 acts as a distinctive enzyme that catalyzes a tandem sugar transfer to the ortho-dihydroxy group on the caffeoyl moiety to form the unusual ortho-diglucosylated product, while LbUGT1 accurately discriminates caffeoyl and dihydrocaffeoyl groups to catalyze a site-selective sugar transfer. Crystal structure analysis of the complexes of LbUGT1 and LbUGT3 with UDP, combined with molecular dynamics simulations, revealed the structural basis of the difference in glycosylation selectivity between LbUGT1 and LbUGT3. Site-directed mutagenesis illuminates a conserved tyrosine residue (Y389 in LbUGT1 and Y390 in LbUGT3) in PSPG box that plays a crucial role in regulating the regioselectivity of LbUGT1 and LbUGT3. Our study thus sheds light on the enzymatic underpinnings of the chemical diversity of lycibarbarspermidines in wolfberry, and expands the repertoire of glycosyltransferases in nature.
Assuntos
Glicosiltransferases , Lycium , Glicosiltransferases/metabolismo , Glicosiltransferases/química , Glicosiltransferases/genética , Glicosilação , Lycium/enzimologia , Lycium/metabolismo , Lycium/química , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/química , Glicosídeos/metabolismo , Glicosídeos/química , Cristalografia por Raios X , Piperidinas/metabolismo , Piperidinas/química , Especificidade por SubstratoRESUMO
Bio-assay guided fractionation of the methanolic extract of Aloe vera resin and Lycium shawii stem successively afforded twenty three compounds; fourteen (1-14) from A. vera and nine (15-23) from L. shawii. All these compounds were characterized by 1D and 2D NMR spectroscopic techniques viz., 1H, 13C, DEPT, HSQC, HMBC, and COSY, and NEOSY, ESI-MS and compared with the reported literature. These compounds were assessed for their potential as urease inhibitors targeted in peptic ulcer. Among crude extracts and fractions of A. vera resin, n-butanol fraction (23.5⯱â¯1.7⯵g·mL-1) showed the most potent urease inhibition followed by methanol (30.9⯱â¯0.3⯵g/mL) and ethyl acetate (31.7⯱â¯0.5⯵g·mL-1). In case of L. shawii, ethyl acetate fraction exhibited the highest urease activity (41.0⯱â¯1.4⯵g/mL) trailed by dichloromethane (55.2⯱â¯1.5⯵g/mL) fraction. Among the isolates, compounds 7, 11 and 23 were found to be excellent urease inhibitors with IC50 values of 14.5⯱â¯0.90⯵M, (16.7⯱â¯0.16⯵M) and 14.0⯱â¯0.8⯵M, respectively. To the best of our knowledge, this is the first report on the urease enzyme inhibitory activity of the said compounds excluding compound 18. In addition, the urease activity of different fractions of L. shawii stem was also reported for the first time. The molecular docking studies showed that all the active compounds well accommodate in the active site of the urease enzyme by interacting with key amino acids.
Assuntos
Aloe/química , Produtos Biológicos/farmacologia , Inibidores Enzimáticos/farmacologia , Extratos Vegetais/farmacologia , Resinas Vegetais/química , Urease/antagonistas & inibidores , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Lycium/enzimologia , Simulação de Acoplamento Molecular , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Relação Estrutura-Atividade , Urease/metabolismoRESUMO
Cortex Lycii (root back of Lycium chinense) has is a famous traditional Chinese medicine which displays several pharmacological activities including antioxidant and antidiabetic properties. We investigated the effect of the ethyl acetate fraction (QCL) of Cortex Lycii on the enzymes involved in the metabolism of carbohydrate in diabetic rat models. Streptozotocin-nicotinamide (110 and 65mg/kg body weight, respectively) was used to induce diabetes. Diabetic rats were treated with QCL (100, 200 and 400 mg/kg) and glibenclamide (600 µg/kg) daily for six weeks. Upon the completion of treatment, fasting blood glucose (FBG), insulin, glycosylated haemoglobin (HbA1c), haemoglobin (Hb), hexokinase, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase levels were measured by biochemical assays. Likewise, the body weight, food and water intake was monitored and measured. Diabetic rats displayed significant elevation in the blood glucose, glycosylated hemoglobin and a marked decrease in plasma insulin and hemoglobin. Furthermore, the levels of key enzymes including fructose-1,6-bisphosphatase, glucose-6-phosphatase phosphoenolpyruvate carboxykinase were significantly increased while the activity levels of hexokinase, glucose-6-phosphate dehydrogenase and glycogen were significantly down regulated in diabetic rats. However, treatment of diabetic rats with Cortex Lycii led to a significant reduction the FGB, food and water intake and an increase in the plasma insulin level. Treatment with Cortex Lycii also reversed the altered activity profiles of the key enzymes mentioned above in a dose dependent manner. Our results suggested that Cortex Lycii has a promising therapeutic option in the management of diabetic complications relating to glucose homeostasis and carbohydrate metabolism.
Assuntos
Glicemia/efeitos dos fármacos , Metabolismo dos Carboidratos/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Enzimas/farmacologia , Hipoglicemiantes/farmacologia , Lycium/enzimologia , Niacinamida , Extratos Vegetais/farmacologia , Estreptozocina , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Relação Dose-Resposta a Droga , Enzimas/isolamento & purificação , Hemoglobinas Glicadas/metabolismo , Hipoglicemiantes/isolamento & purificação , Insulina/sangue , Masculino , Extratos Vegetais/isolamento & purificação , Ratos Sprague-DawleyRESUMO
As compatible solute, glycine betaine (GB) plays a significant role in salinity tolerance in GB accumulating plants. Solanaceous crops such as tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum) are salt sensitive and naturally GB non-accumulators. In Solanaceae, only the Lycium genus has been recorded as halophytes in China, and several Lycium species have been reported as GB accumulators. The last biosynthetic step of GB is catalyzed by aminoaldehyde dehydrogenase (AMADH) with betaine aldehyde dehydrogenase (BADH) activities. Failure of GB synthesis in tomato and tobacco was attributed to lack of BADH activity. Here, by comparing the BADH functional residues of AMADHs between the Lycium genus and solanaceous crops, we predict that all studied AMADH1s have low BADH activities while only LbAMADH2 from L. barbarum has high BADH activity. For two AMADHs in L. ruthenicum, results from substrate enzyme assays confirmed low BADH activity of LrAMADH1 and no BADH activity of LrAMADH2. Despite the very low GB contents in L. ruthenicum seedlings (< 0.5 µmol g-1 fresh weight), GB contents in fruits are up to 150 µmol g-1 FW, inferring fruits of L. ruthenicum as good GB sources. In NaCl treated seedlings, accompanied by elevated GB accumulation, expression of LrAMADH1 was up-regulated, indicating response of LrAMADH1 to salt stress in L. ruthenicum. Virus-induced silence of LrAMADH1 leads to less GB accumulation than control, revealing that LrAMADH1 participates in GB synthesis in planta. Collectively, our results show that LrAMADH1 is the bona fide BADH, which responds to salt stress in L. ruthenicum.
Assuntos
Betaína-Aldeído Desidrogenase/metabolismo , Lycium/enzimologia , Betaína-Aldeído Desidrogenase/genética , Lycium/genética , Lycium/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tolerância ao Sal , Plântula/enzimologia , Plântula/genética , Plântula/fisiologiaRESUMO
Flavonoids, as plant secondary metabolites, are widespread throughout the plant kingdom and involved in many physiological and biochemical processes. Drought resistance is attributed to flavonoids with respect to protective functions in the cell wall and membranes. The flavanone 3-hydroxylase (F3H) gene which encodes flavanone 3-hydroxylase, is essential in flavonoids biosynthetic pathway. Lycium chinense (L. chinense) is a deciduous woody perennial halophyte that grows under a large variety of environmental conditions and survives under extreme drought stress. A novel cDNA sequence coding a F3H gene in Lycium chinense (LcF3H, GenBank: KJ636468.1) was isolated. The open reading frame of LcF3H comprised 1101 bp encoding a polypeptide of 366 amino acids with a molecular weight of about 42 kDa and an isoelectric point of 5.32. The deduced LcF3H protein showed high identities with other plant F3Hs, and the conserved motifs were found in LcF3H at similar positions like other F3Hs. The recombinant protein converted naringen into dihydrokaempferol in vitro. Since studies have shown that amongst flavonoids, flavan-3-ols (catechin and epicatechin) have direct free radical scavenging activity to maintain the normal physiological function of cells in vivo, these data support the possible relationship between the oxidative damage and the regulation of LcF3H gene expression in L. chinense under drought stress. In order to better understand the biotechnological potential of LcF3H, gene overexpression was conducted in tobacco. The content of flavan-3-ols and the tolerance to drought stress were increased in LcF3H overexpressing tobacco. Analysis of transgenic tobacco lines also showed that antioxidant enzyme activities were increased meanwhile the malondialdehyde (MDA) content and the content of H2O2 were reduced comparing to nontransformed tobacco plants. Furthermore, the photosynthesis rate was less decreased in the transgenetic plants. These results suggest that LcF3H plays a role in enhancing drought tolerance in L. chinense, and its overexpression increases tolerance to drought stress by improving the antioxidant system in tobacco.
Assuntos
Ácido Abscísico/metabolismo , Regulação da Expressão Gênica de Plantas , Lycium/enzimologia , Oxigenases de Função Mista/genética , Reguladores de Crescimento de Plantas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Antioxidantes/metabolismo , Vias Biossintéticas , Clonagem Molecular , Secas , Flavonoides/metabolismo , Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Lycium/genética , Lycium/fisiologia , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Fotossíntese , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Alinhamento de Sequência , Estresse Fisiológico , Nicotiana/enzimologia , Nicotiana/genética , Nicotiana/fisiologiaRESUMO
Lutein plays an important role in protecting the photosynthetic apparatus from photodamage and eliminating ROS to render normal physiological function of cells. As a rate-limiting step for lutein synthesis in plants, lycopene ε-cyclase catalyzes lycopene to δ-carotene. We cloned a lycopene ε-cyclase gene (Lcε-LYC) from Lycium chinense (L. chinense), a deciduous woody perennial halophyte growing in various environmental conditions. The Lcε-LYC gene has an ORF of 1569bp encoding a protein of 522 aa. The deduced amino acid sequence of Lcε-LYC gene has higher homology with LycEs in other plants, such as Nicotiana tabacum and Solanum tuberosum. When L. chinense was exposed to chilling stress, relative expression of Lcε-LYC increased. To study the protective role of Lcε-LYC against chilling stress, we overexpressed the Lcε-LYC gene in Arabidopsis thaliana. Lcε-LYC overexpression led to an increase of lutein accumulation in transgenic A. thaliana, and the content of lutein decreased when transgenics were under cold conditions. In addition, the transgenic plants under chilling stress displayed higher activities of superoxide dismutase (SOD) and peroxidase (POD) and less H2O2 and malondialdehyde (MDA) than the control. Moreover, the photosynthesis rate, photosystem II activity (Fv/fm), and Non-photochemical quenching (NPQ) also increased in the transgenetic plants. On the whole, overexpression of Lcε-LYC ameliorates photoinhibition and photooxidation, and decreases the sensitivity of photosynthesis to chilling stress in transgenic plants.
Assuntos
Adaptação Fisiológica/genética , Arabidopsis/enzimologia , Temperatura Baixa , Liases Intramoleculares/genética , Lycium/enzimologia , Arabidopsis/genética , Arabidopsis/fisiologia , Genes de Plantas , Peróxido de Hidrogênio/metabolismo , Malondialdeído/metabolismo , Fotossíntese , Plantas Geneticamente ModificadasAssuntos
Genes de Plantas , Lycium/enzimologia , Lycium/genética , Oxirredutases/genética , Sequência de Aminoácidos , Carotenoides/metabolismo , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Oxirredutases/química , Oxirredutases/metabolismo , Filogenia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie , Estresse Fisiológico/genéticaRESUMO
KEY MESSAGE: A GSHS gene, LcGSHS , was cloned from L. chinense for the first time. Evidence is presented here that endogenous SA accumulation maybe important for the regulation of LcGSHS expression level. Glutathione (GSH) plays a pivotal role in heavy metal detoxification. GSH synthetase (GSHS) catalyzes the rate-limiting step of GSH synthesis in plants. Salicylic acid (SA) is one of the important plant hormones, which plays a critical role in triggering plant responses to different stresses such as cadmium (Cd) stress. Until now, little has been done to explore the relationship among the accumulation of endogenous SA, GSHS transcript levels and the GSH content in plants under Cd treatment and we will investigate this link in this study. The chlorophyll content, transcripts level of LcGSHS gene, endogenous SA accumulation, GSH accumulation and Cd concentration in the leaves of Lycium chinense were studied under different treatment conditions. Endogenous SA, LcGSHS transcript expression and GSH content can be induced by Cd treatment in L. chinense, however, reduced by co-treatment with 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of SA biosynthesis. Strong staining was observed in the leaves of Arabidopsis expressing ProLcGSHS::GUS under Cd stress and the staining was reduced by co-treatment with AIP. The transgenic Arabidopsis expressing ProLcGSHS::LcGSHS also showed greater tolerance to Cd stress than wild types. Evidence was presented here that under Cd stress, GSH accumulation occurred via enhanced LcGSHS gene expression and the SA signaling cascade was involved in this accumulation. Furthermore, the overexpression of LcGSHS in transgenic Arabidopsis resulted in greater tolerance to Cd stress than wild-type lines.
Assuntos
Arabidopsis/fisiologia , Cádmio/toxicidade , Regulação da Expressão Gênica de Plantas , Glutationa Sintase/genética , Lycium/enzimologia , Ácido Salicílico/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Arabidopsis/genética , Glutationa Sintase/metabolismo , Lycium/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Estresse FisiológicoRESUMO
Violaxanthin de-epoxidase (VDE) plays an important role in protecting the photosynthetic apparatus from photo-damage by dissipating excessively absorbed light energy as heat, via the conversion of violaxanthin (V) to intermediate product antheraxanthin (A) and final product zeaxanthin (Z) under light stress. We have cloned a VDE gene (LcVDE) from Lycium chinense, a deciduous woody perennial halophyte, which can grow in a large variety of soil types. The amino acid sequence of LcVDE has high homology with VDEs in other plants. Under drought stress, relative expression of LcVDE and the de-epoxidation ratio (Z+0.5A)/(V+A+Z) increased rapidly, and non-photochemical quenching (NPQ) also rose. Interestingly, these elevations induced by drought stress were reduced by the topical administration of abamine SG, a potent ABA inhibitor via inhibition of NCED in the ABA synthesis pathway. Until now, little has been done to explore the relationship between endogenous ABA and the expression of VDE genes. Since V serves as a common precursor for ABA, these data support the possible involvement of endogenous ABA in the positive feedback regulation of LcVDE gene expression in L. chinense under drought stress. Moreover, the LcVDE may be involved in modulating the level of photosynthesis damage caused by drought stress. Furthermore, the ratio of (Z+0.5A)/(V+A+Z) and NPQ increased more in transgenic Arabidopsis over-expressing LcVDE gene than the wild types under drought stress. The maximum quantum yield of primary photochemistry of PSII (Fv/Fm) in transgenic Arabidopsis decreased more slowly during the stressed period than that in wild types under the same conditions. Furthermore, transgenic Arabidopsis over-expressing LcVDE showed increased tolerance to drought stress.
Assuntos
Arabidopsis/fisiologia , Retroalimentação Fisiológica , Lycium/genética , Oxirredutases/genética , Reguladores de Crescimento de Plantas/metabolismo , Ácido Abscísico/metabolismo , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Sequência de Bases , Secas , Regulação da Expressão Gênica de Plantas , Luz , Lycium/enzimologia , Dados de Sequência Molecular , Oxirredutases/metabolismo , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Alinhamento de Sequência , Análise de Sequência de DNA , Estresse Fisiológico , Xantofilas/metabolismoRESUMO
Carotenoids are key precursor for aroma compounds in plants. Although the fruit of Lycium chinense contains numerous carotenoids, the formation mechanism of aroma compounds in L. chinense is still poorly understood. In this study, a new carotenoid cleavage dioxygenase (termed LmCCD1) was identified from the leaves of L. chinense. Expression analysis by semiquantitative PCR reveals that LmCCD1 gene is expressed in different tissues of L. chinense, and dominant expression of LmCCD1 gene was found in leaves, flowers, and ripe fruits. In addition, the expression level of LmCCD1 in fruits is in accordance with the content of ß-ionone. Finally, recombinantly expressed LmCCD1 can cleave ß-carotene and lycopene to produce ß-ionone and pseudoionone in in vitro assays. These results indicate that LmCCD1 a novel carotenoids cleavage dioxygenase gene that may regulate the metabolic pathways responsible for aroma metabolite production (such as ß-ionone and pseudoionone) in L. chinense has been identified.
Assuntos
Dioxigenases/genética , Dioxigenases/metabolismo , Lycium/enzimologia , Lycium/genética , Sequência de Aminoácidos , Clonagem Molecular , Dioxigenases/química , Dioxigenases/isolamento & purificação , Regulação da Expressão Gênica de Plantas , Lycium/metabolismo , Dados de Sequência Molecular , Compostos Orgânicos Voláteis/metabolismoRESUMO
BACKGROUND: The traditional Chinese medicinal plants Lycium barbarum L. and L. ruthenicum Murr. are valued for the abundance of bioactive carotenoids and anthocyanins in their fruits, respectively. However, the cellular and molecular mechanisms contributing to their species-specific bioactive profiles remain poorly understood. RESULTS: In this study, the red fruit (RF) of L. barbarum was found to accumulate high levels of carotenoids (primarily zeaxanthin), while they were undetectable in the black fruit (BF) of L. ruthenicum. Cytological and gene transcriptional analyses revealed that the chromoplast differentiation that occurs in the chloroplast during fruit ripening only occurs in RF, indicating that the lack of chromoplast biogenesis in BF leads to no sink for carotenoid storage and the failure to synthesize carotenoids. Similar enzyme activities of phytoene synthase 1 (PSY1), chromoplast-specific lycopene ß-cyclase (CYC-B) and ß-carotene hydroxylase 2 (CRTR-B2) were observed in both L. ruthenicum and L. barbarum, suggesting that the undetectable carotenoid levels in BF were not due to the inactivation of carotenoid biosynthetic enzymes. The transcript levels of the carotenoid biosynthetic genes, particularly PSY1, phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), CYC-B and CRTR-B2, were greatly increased during RF ripening, indicating increased zeaxanthin biosynthesis. Additionally, carotenoid cleavage dioxygenase 4 (CCD4) was expressed at much higher levels in BF than in RF, suggesting continuous carotenoid degradation in BF. CONCLUSIONS: The failure of the chromoplast development in BF causes low carotenoid biosynthesis levels and continuous carotenoid degradation, which ultimately leads to undetectable carotenoid levels in ripe BF. In contrast, the successful chromoplast biogenesis in RF furnishes the sink necessary for carotenoid storage. Based on this observation, the abundant zeaxanthin accumulation in RF is primarily determined via both the large carotenoid biosynthesis levels and the lack of carotenoid degradation, which are regulated at the transcriptional level.
Assuntos
Carotenoides/metabolismo , Frutas/química , Lycium/química , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Antocianinas/metabolismo , Frutas/enzimologia , Frutas/genética , Regulação da Expressão Gênica de Plantas , Liases Intramoleculares/genética , Liases Intramoleculares/metabolismo , Lycium/enzimologia , Lycium/genética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Folhas de Planta/química , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Medicinais , Plastídeos/metabolismo , Especificidade da EspécieRESUMO
Lycium barbarum contains high levels of zeaxanthin, which is produced by the conversion of ß-carotene into zeaxanthin. ß-Carotene hydroxylase catalyzes this reaction. We cloned a cDNA (chyb) encoding ß-carotene hydroxylase (Chyb) from the L. barbarum leaf. A 939-bp full-length cDNA sequence was determined with 3'-rapid amplification of cDNA end assay encoding a deduced Chyb protein (34.8 kDa) with a theoretical isoelectric point of 8.36. A bioinformatics analysis showed that the L. barbarum Chyb was located in the chloroplast. Further, to investigate the catalytic activity of the L. barbarum Chyb, a complementation analysis was conducted in Escherichia coli. The results strongly demonstrated that Chyb can catalyze ß-carotene to produce zeaxanthin. Thus, this study suggests that L. barbarum ß-carotene hydroxylase could be a means of zeaxanthin production by genetic manipulation in E. coli.
Assuntos
Clonagem Molecular , Lycium/genética , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , DNA Complementar , Escherichia coli , Regulação da Expressão Gênica de Plantas , Lycium/enzimologia , Oxigenases de Função Mista/isolamento & purificação , Zeaxantinas/metabolismo , beta Caroteno/genética , beta Caroteno/metabolismoRESUMO
Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 4'-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4'-O-glucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49% identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (+) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC-MS and HPLC, which confirmed its specificity for genistein 4'-O-glucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatio-temporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism.
Assuntos
Bacopa/enzimologia , Benzopiranos/metabolismo , Expressão Gênica , Glicosiltransferases/química , Proteínas de Plantas/química , Motivos de Aminoácidos , Bacopa/classificação , Bacopa/efeitos dos fármacos , Bacopa/genética , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Índia , Lycium/química , Lycium/enzimologia , Dados de Sequência Molecular , Filogenia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Caules de Planta/efeitos dos fármacos , Caules de Planta/enzimologia , Caules de Planta/genética , Plantas Medicinais , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ácido Salicílico/farmacologia , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Over 50 years ago, Baker (1955, 1967) suggested that self-compatible species were more likely than self-incompatible species to establish new populations on oceanic islands. His logic was straightforward and rested on the assumption that colonization was infrequent; thus, mate limitation favored the establishment of self-fertilizing individuals. In support of Baker's rule, many authors have documented high frequencies of self-compatibility on islands, and recent work has solidified the generality of Baker's ideas. The genus Lycium (Solanaceae) has ca. 80 species distributed worldwide, and phylogenetic studies suggest that Lycium originated in South America and dispersed to the Old World a single time. Previous analyses of the S-RNase gene, which controls the stylar component of self-incompatibility, have shown that gametophytically controlled self-incompatibility is ancestral within the genus, making Lycium a good model for investigating Baker's assertions concerning reproductive assurance following oceanic dispersal. Lycium is also useful for investigations of reproductive evolution, given that species vary both in sexual expression and the presence of self-incompatibility. A model for the evolution of gender dimorphism suggests that polyploidy breaks down self-incompatibility, leading to the evolution of gender dimorphism, which arises as an alternative outcrossing mechanism. There is a perfect association of dimorphic gender expression, polyploidy, and self-compatibility (vs. cosexuality, diploidy, and self-incompatibility) among North American Lycium. Although the association between ploidy level and gender expression also holds for African Lycium, to date no studies of mating systems have been initiated in Old World species. Here, using controlled pollinations, we document strong self-incompatibility in two cosexual, diploid species of African Lycium. Further, we sequence the S-RNase gene in 15 individuals from five cosexual, diploid species of African Lycium and recover 24 putative alleles. Genealogical analyses indicate reduced trans-generic diversity of S-RNases in the Old World compared to the New World. We suggest that genetic diversity at this locus was reduced as a result of a founder event, but, despite the bottleneck, self-incompatibility was maintained in the Old World. Maximum-likelihood analyses of codon substitution patterns indicate that positive Darwinian selection has been relatively strong in the Old World, suggesting the rediversification of S-RNases following a bottleneck. The present data thus provide a dramatic exception to Baker's rule, in addition to supporting a key assumption of the Miller and Venable (2000) model, namely that self-incompatibility is associated with diploidy and cosexuality.
Assuntos
Lycium/genética , Lycium/fisiologia , Cruzamento , Evolução Molecular , Efeito Fundador , Variação Genética , Genótipo , Geografia , Lycium/classificação , Lycium/enzimologia , Dados de Sequência Molecular , Filogenia , Polinização/fisiologia , Ribonucleases/genética , Seleção GenéticaRESUMO
We characterized allelic diversity at the locus controlling self-incompatibility (SI) for a population of Lycium parishii (Solanaceae) from Organ Pipe National Monument, Arizona. Twenty-four partial sequences of S-RNase alleles were recovered from 25 individuals. Estimates of allelic diversity range from 23 to 27 alleles and, consistent with expectations for SI, individuals are heterozygous. We compare S-RNase diversity, patterns of molecular evolution, and the genealogical structure of alleles from L. parishii to a previously studied population of its congener L. andersonii. Gametophytic SI is well characterized for Solanaceae and although balancing selection is hypothesized to be responsible for high levels of allelic divergence, the pattern of selection varies depending on the portion of the gene considered. Site-specific models investigating patterns of selection for L. parishii and L. andersonii indicate that positive selection occurs in those regions of the S-RNase gene hypothesized as important to the recognition response, whereas positive selection was not detected for any position within regions previously characterized as conserved. A 10-species genealogy including S-RNases from a pair of congeners from each of five genera in Solanaceae reveals extensive transgeneric evolution of L. parishii S-RNases. Further, within Lycium, the Dn/Ds ratios for pairs of closely related alleles for intraspecific versus interspecific comparisons were not significantly different, suggesting that the S-RNase diversity recovered in these two species was present prior to the speciation event separating them. Despite this, two S-RNases from L. parishii are identical to two previously reported alleles for L. andersonii, suggesting gene flow between these species.