RESUMEN
BACKGROUND: Phenylpropanoids represent a diverse class of industrially important secondary metabolites, synthesized in plants from phenylalanine and tyrosine. Cyanobacteria have a great potential for sustainable production of phenylpropanoids directly from CO2, due to their photosynthetic lifestyle with a fast growth compared to plants and the ease of generating genetically engineered strains. This study focuses on photosynthetic production of the starting compounds of the phenylpropanoid pathway, trans-cinnamic acid and p-coumaric acid, in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). RESULTS: A selected set of phenylalanine ammonia lyase (PAL) enzymes from different organisms was overexpressed in Synechocystis, and the productivities of the resulting strains compared. To further improve the titer of target compounds, we evaluated the use of stronger expression cassettes for increasing PAL protein levels, as well as knock-out of the laccase gene slr1573, as this was previously reported to prevent degradation of the target compounds in the cell. Finally, to investigate the effect of growth conditions on the production of trans-cinnamic and p-coumaric acids from Synechocystis, cultivation conditions promoting rapid, high density growth were tested. Comparing the different PALs, the highest specific titer was achieved for the strain AtC, expressing PAL from Arabidopsis thaliana. A subsequent increase of protein level did not improve the productivity. Production of target compounds in strains where the slr1573 laccase had been knocked out was found to be lower compared to strains with wild type background, and the Δslr1573 strains exhibited a strong phenotype of slower growth rate and lower pigment content. Application of a high-density cultivation system for the growth of production strains allowed reaching the highest total titers of trans-cinnamic and p-coumaric acids reported so far, at around 0.8 and 0.4 g L-1, respectively, after 4 days. CONCLUSIONS: Production of trans-cinnamic acid, unlike that of p-coumaric acid, is not limited by the protein level of heterologously expressed PAL in Synechocystis. High density cultivation led to higher titres of both products, while knocking out slr1573 did not have a positive effect on production. This work contributes to capability of exploiting the primary metabolism of cyanobacteria for sustainable production of plant phenylpropanoids.
Asunto(s)
Cinamatos/metabolismo , Ácidos Cumáricos/metabolismo , Ingeniería Metabólica , Fenilanina Amoníaco-Liasa/biosíntesis , Fenilanina Amoníaco-Liasa/genética , Synechocystis/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Expresión Génica , Fenilanina Amoníaco-Liasa/metabolismo , Fotosíntesis , Synechocystis/genética , Synechocystis/crecimiento & desarrolloRESUMEN
Plant growth promoting rhizobacteria (PGPR) are found to control the plant diseases by adopting various mechanisms. Induced systemic resistance (ISR) is an important defensive strategy manifested by plants against numerous pathogens especially infecting at aerial parts. Rhizobacteria elicit ISR by inducing different pathways in plants through production of various metabolites. In the present study, potential of Bacillus spp. KFP-5, KFP-7, KFP-17 was assessed to induce antioxidant enzymes against Pyricularia oryzae infection in rice. The antagonistic Bacillus spp. significantly induced antioxidant defense enzymes i-e superoxide dismutase (1.7-1.9-fold), peroxidase (3.5-4.1-fold), polyphenol oxidase (3.0-3.8-fold), phenylalanine ammonia-lyase (3.9-4.4-fold), in rice leaves and roots under hydroponic and soil conditions respectively. Furthermore, the antagonistic Bacillus spp significantly colonized the rice plants (2.0E+00-9.1E+08) and secreted multiple biocontrol determinants like protease (1.1-5.5 U/mg of soil or U/mL of hydroponic solution), glucanase, (1.0-1.3 U/mg of soil or U/mL of hydroponic solution), siderophores (6.5-42.8 µg/mL or mg) in the rhizosphere of different rice varieties. The results showed that treatment with Bacillus spp. enhanced the antioxidant defense activities in infected rice, thus alleviating P. oryzae induced oxidative damage and suppressing blast disease incidence.
Asunto(s)
Antioxidantes/metabolismo , Bacillus/metabolismo , Resistencia a la Enfermedad/genética , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Ascomicetos/patogenicidad , Catecol Oxidasa/biosíntesis , Oryza/crecimiento & desarrollo , Peroxidasa/biosíntesis , Fenilanina Amoníaco-Liasa/biosíntesis , Hojas de la Planta/enzimología , Hojas de la Planta/metabolismo , Raíces de Plantas/enzimología , Raíces de Plantas/microbiología , Superóxido Dismutasa/biosíntesisRESUMEN
Phenylalanine ammonia-lyase (PAL), the branch point enzyme controlling the flow of primary metabolism into second metabolism, converts the L-phenylalanine (L-Phe) to yield cinnamic acid. Based on the sequencing data available from eight transcriptome projects, six PAL genes have been screened out, cloned, and designated as CsPALa-CsPALf. The phylogenetic tree showed that CsPALs were divided into three subgroups, PALa and PALb, PALc and PALd, and PALe and PALf. All six CsPALs exhibited indiscriminate cytosolic locations in epidermis cells and mesophyll cells. Then, the expression profiles of six PAL genes were qualitatively investigated and they displayed tissue-/induced-expression specificity in several tissues or under different exogenous treatments. Furthermore, in vitro enzymatic assays showed that all six recombinant proteins were characterized by the strict substrate specificity toward L-Phe, but no activity toward L-Tyr, and they displayed subtle differences in kinetics and enzymatic properties. These results indicate that CsPALs play both distinct and overlapping roles in plant growth and responses to environmental cues.
Asunto(s)
Camellia sinensis/enzimología , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Fenilanina Amoníaco-Liasa/biosíntesis , Filogenia , Proteínas de Plantas/biosíntesis , Camellia sinensis/genética , Clonación Molecular , Perfilación de la Expresión Génica , Fenilanina Amoníaco-Liasa/genética , Proteínas de Plantas/genéticaRESUMEN
Rhodotorula glutinis is capable of synthesizing numerous valuable compounds with a wide industrial usage. Biomass of this yeast constitutes sources of microbiological oils, and the whole pool of fatty acids is dominated by oleic, linoleic, and palmitic acid. Due to its composition, the lipids may be useful as a source for the production of the so-called third-generation biodiesel. These yeasts are also capable of synthesizing carotenoids such as ß-carotene, torulene, and torularhodin. Due to their health-promoting characteristics, carotenoids are commonly used in the cosmetic, pharmaceutical, and food industries. They are also used as additives in fodders for livestock, fish, and crustaceans. A significant characteristic of R. glutinis is its capability to produce numerous enzymes, in particular, phenylalanine ammonia lyase (PAL). This enzyme is used in the food industry in the production of L-phenylalanine that constitutes the substrate for the synthesis of aspartame-a sweetener commonly used in the food industry.
Asunto(s)
Carotenoides/biosíntesis , Enzimas/química , Ácidos Grasos/biosíntesis , Microbiología Industrial , Rhodotorula/química , Biocombustibles/microbiología , Biomasa , Ácido Linoleico/biosíntesis , Ácido Oléico/biosíntesis , Ácido Palmítico/metabolismo , Fenilalanina/metabolismo , Fenilanina Amoníaco-Liasa/biosíntesis , Rhodotorula/enzimología , beta Caroteno/biosíntesisRESUMEN
BACKGROUND: Recently, an increase of interest in the modification of food products on each step of production (breeding, production technology, storage condition) is observed. Nutritional properties as well as level and activity of bioactive compounds in plant-origin food may be modified using a range of technological and biotechnological practices and elicitation should be mentioned between them. METHODS: Elicitation with willow bark infusion supported by feeding with the phenylpropanoid pathway precursors were used for improving the quality of buckwheat sprouts. Special emphasis has been placed on the metabolomic and biochemical changes and the mechanism of overproduction of low-molecular antioxidants. RESULTS: The accumulation of phenolics is caused by stimulation of two main enzymes the phenylpropanoid pathway (tyrosine ammonia-lyase and phenylalanine ammonia-lyase). Tyrosine ammonia-lyase activities were effectively induced by feeding with tyrosine (about four times that of the control), whereas phenylalanine ammonia-lyase activity was the highest in the elicited control sprouts and those fed with shikimic acid (an increase by 60% compared to the control). Shikimic acid feeding (both elicited and non-elicited sprouts) effectively improved the total phenolics (by about 10% and 20%, respectively), condensed tannins (by about 30% and 28%, respectively), and flavonoids (by about 46% and 70%, respectively). Significant increase of vitexin, rutin, chlorogenic acid and isoorientin contents was also observed. The treatments increased the ascorbic acid content, too. Total antioxidant capacity of sprouts was most effectively increased by feeding with shikimic acid and further elicitation. CONCLUSIONS: The studies transfer biotechnology commonly used for the induction of overproduction of secondary metabolites in plant cell line systems to low-processed food production. The obtained results could be used for better understanding of the effect of elicitation and precursor feeding on antioxidants production and contribute to improving the buckwheat sprouts quality.
Asunto(s)
Amoníaco-Liasas/biosíntesis , Antioxidantes/metabolismo , Fagopyrum/metabolismo , Flavonoides/biosíntesis , Fenilanina Amoníaco-Liasa/biosíntesis , Plantones/metabolismo , Ácido Shikímico/metabolismo , Agroquímicos/metabolismo , Amoníaco-Liasas/química , Antioxidantes/análisis , Antioxidantes/química , Ácido Ascórbico/análisis , Ácido Ascórbico/biosíntesis , Ácido Clorogénico/análisis , Ácido Clorogénico/metabolismo , Inducción Enzimática , Fagopyrum/química , Fagopyrum/crecimiento & desarrollo , Flavonoides/análisis , Calidad de los Alimentos , Alimentos Orgánicos/análisis , Hidroponía , Peso Molecular , Fenilanina Amoníaco-Liasa/química , Corteza de la Planta/química , Extractos Vegetales/metabolismo , Proteínas de Plantas/agonistas , Proteínas de Plantas/biosíntesis , Polonia , Proantocianidinas/análisis , Proantocianidinas/biosíntesis , Salix/química , Plantones/química , Plantones/crecimiento & desarrollo , Tirosina/metabolismoRESUMEN
Phenylalanine ammonia lyase (PAL) and p-coumarate 3-hydroxylase (C3H) are key enzymes in the phenylpropanoid pathway. The relative expression of PAL and C3H was evaluated in mango fruit cultivar 'Ataulfo' in four ripening stages (RS1, RS2, RS3, and RS4) by quantitative polymerase chain reaction. In addition, enzyme activity of PAL and C3H was determined in mango fruits during ripening. The PAL levels were downregulated at the RS2 and RS3 stages, while C3H levels were upregulated in fruits only at RS3. The enzyme activity of PAL followed a pattern that was different from that of the PAL expression, thus suggesting regulation at several levels. For C3H, a regulation at the transcriptional level is suggested because a similar pattern was revealed by its activity and transcript level. In this study, the complexity of secondary metabolite biosynthesis regulation is emphasized because PAL and C3H enzymes are involved in the biosynthesis of several secondary metabolites that are active during all mango ripening stages.
Asunto(s)
Frutas/crecimiento & desarrollo , Mangifera/genética , Oxigenasas de Función Mixta/biosíntesis , Fenilanina Amoníaco-Liasa/biosíntesis , Clonación Molecular , Frutas/genética , Regulación de la Expresión Génica de las Plantas , Mangifera/crecimiento & desarrollo , Oxigenasas de Función Mixta/genética , Fenilanina Amoníaco-Liasa/genética , Reacción en Cadena de la PolimerasaRESUMEN
Safflower (Carthamus tinctorius L.) serves as a reference dicot for investigation of defence mechanisms in Asteraceae due to abundant secondary metabolites and high resistance/tolerance to environmental stresses. In plants, phenylpropanoid and flavonoid pathways are considered as two central defence signalling cascades in stress conditions. Here, we describe the isolation of two major genes in these pathways, CtPAL (phenylalanine ammonia-lyase) and CtCHS (chalcone synthase) in safflower along with monitoring their expression profiles in different stress circumstances. The aa (amino acid) sequence of isolated region of CtPAL possesses the maximum identity up to 96% to its orthologue in Cynara scolymus, while that of CtCHS retains the highest identity to its orthologue in Callistephus chinensis up to 96%. Experiments for gene expression profiling of CtPAL and CtCHS were performed after the treatment of seedlings with 0.1 and 1 mM SA (salicylic acid), wounding and salinity stress. The results of semi-quantitative RT-PCR revealed that both CtPAL and CtCHS genes are further responsive to higher concentration of SA with dissimilar patterns. Regarding wounding stress, CtPAL gets slightly induced upon injury at 3 hat (hours after treatment) (hat), whereas CtCHS gets greatly induced at 3 hat and levels off gradually afterward. Upon salinity stress, CtPAL displays a similar expression pattern by getting slightly induced at 3 hat, but CtCHS exhibits a biphasic expression profile with two prominent peaks at 3 and 24 hat. These results substantiate the involvement of phenylpropanoid and particularly flavonoid pathways in safflower during wounding and especially salinity stress.
Asunto(s)
Aciltransferasas/biosíntesis , Carthamus tinctorius/enzimología , Presión Osmótica/fisiología , Fenilanina Amoníaco-Liasa/biosíntesis , Proteínas de Plantas/biosíntesis , Ácido Salicílico/metabolismo , Estrés Fisiológico/fisiología , Flavonoides/biosíntesis , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Transducción de Señal/fisiologíaRESUMEN
The ectomycorrhizal fungus Tricholoma matsutake grows symbiotically with Pinus densiflora. Phenylalanine ammonia-lyase (E.C. 4.3.1.24) catalyzes the conversion of L-phenylalanine to trans-cinnamic acid. The role of fungal phenylalanine ammonia-lyase, however, has not been clear until now. In this study, the gene encoding phenylalanine ammonia-lyase (PAL), which was isolated from T. matsutake, was cloned and characterized. The PAL gene (tmpal) consists of 2,160 nucleotides, coding for a polypeptide containing 719 amino acid residues. The deduced amino acid sequence of tmpal from T. matsutake shows high identity (70%) with that from Laccaria bicolor. Comparative analysis of the PAL genes among T. matsutake and other species of the class Agaricomycetes showed that both active sites and binding sites were significantly conserved among these genes. The transcriptional analysis of the PAL gene revealed a differential gene expression pattern depending on the developmental stages (mycelium, primordium, stipe, pileus, and gills) of T. matsutake. These results suggest that the PAL gene in T. matsutake plays an important role in multiple physiological functions.
Asunto(s)
Fenilanina Amoníaco-Liasa/biosíntesis , Fenilanina Amoníaco-Liasa/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Tricholoma/enzimología , Tricholoma/genética , Dominio Catalítico , Clonación Molecular , Secuencia Conservada , Perfilación de la Expresión Génica , Sistemas de Lectura Abierta , Homología de Secuencia de AminoácidoRESUMEN
The biosynthesis of resveratrol after the application of a precursor for biosynthesis, i.e., phenylalanine (Phe), has been studied. The application of Phe has been shown to increase significantly the expression of the phenylalanine-ammonia-lyase (PAL) and stilbene synthase (STS) genes and enhance the production of resveratrol by 8.5 times. Data on resveratrol production after the addition of Phe and coumaric acid (CA) were compared with known analogs.
Asunto(s)
Células Vegetales/metabolismo , Estilbenos/metabolismo , Vitis/metabolismo , Aciltransferasas/biosíntesis , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Fenilanina Amoníaco-Liasa/biosíntesis , Proteínas de Plantas/biosíntesis , Resveratrol , Vitis/citologíaRESUMEN
BACKGROUND: Since tomato is an important food component, it is imperative to enhance its yield against the activities of many devastating fungal pathogens such as Alternaria alternata. The exploitation of plant innate resistance by cultivation of resistant varieties is an effective measure in this regard. In the present study, 28 tomato varieties were tested against 32 A. alternata isolates, and representative varieties were further evaluated to determine the extent and basis of their antifungal resistance. RESULTS: A significant increase (104.7%) in polyphenols was recorded in the resistant variety Dinaar compared with the susceptible variety Red Tara. Dinaar also exhibited 100% enhancement of alkaloids and terpenoids along with a 30.7% increase in cell wall hemicellulose content. Significant differences were found in physical barriers (cellulose, lignin and pectin) of the representative varieties when stained tissue sections were subjected to colorimetric analysis. Similarly, polyphenol oxidase, peroxidase and phenylalanine ammonia lyase showed increases of 78.37, 114.67 and 125.11% respectively in the resistant variety. Higher expression of glucanase genes was evident from native gel analysis, in which not only the number of isozymes but also the quantity of individual isozymes was significantly increased. CONCLUSION: The resistant variety Dinaar had strong antifungal resistance and can therefore be recommended as suitable for cultivation in the agricultural system of Pakistan.
Asunto(s)
Alternaria/crecimiento & desarrollo , Resistencia a la Enfermedad , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Solanum lycopersicum/microbiología , Alcaloides/biosíntesis , Alternaria/aislamiento & purificación , Alternaria/patogenicidad , Catecol Oxidasa/biosíntesis , Celulosa/biosíntesis , Celulosa/química , Solanum lycopersicum/citología , Solanum lycopersicum/metabolismo , Pakistán , Pectinas/biosíntesis , Peroxidasas/biosíntesis , Fenilanina Amoníaco-Liasa/biosíntesis , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Proteínas de Plantas/biosíntesis , Polifenoles/biosíntesis , Especificidad de la Especie , Terpenos/metabolismo , VirulenciaRESUMEN
The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg(-1) and the k cat/K m was 1.9 × 10(4) mM(-1) s(-1), exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry.
Asunto(s)
Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Fenilanina Amoníaco-Liasa/biosíntesis , Fenilanina Amoníaco-Liasa/genética , Rhodotorula/enzimología , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Metales Pesados , Fenilanina Amoníaco-Liasa/química , Fenilanina Amoníaco-Liasa/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodotorula/genética , Especificidad por Sustrato , Tensoactivos , TemperaturaRESUMEN
The time course of changes in the endogenous content of salicylic acid, the ratio between the acid's free and bound forms, and changes in the activities of phenylalanine ammonia-lyase and catalase in wheat seedling roots under the effect of lectins of two strains of the associative nitrogen-fixing bacterium Azospirillum (A. brasilense Sp7 and its mutant defective in lectin activity, A. brasilense Sp7.2.3) is investigated. Differences in plant response to the action of the lectins from these two strains are established. On the basis of the obtained data, a model is proposed for lectin-assisted induction of resistance, according to which the lectin effect on the roots of seedlings results in the accumulation of free salicylic acid, which inhibits catalase activity, ultimately leading to accumulation of hydrogen peroxide and formation of induced resistance.
Asunto(s)
Azospirillum/metabolismo , Ácido Salicílico/metabolismo , Plantones/metabolismo , Triticum/metabolismo , Catalasa/biosíntesis , Peróxido de Hidrógeno/metabolismo , Lectinas/metabolismo , Fijación del Nitrógeno/genética , Fenilanina Amoníaco-Liasa/biosíntesis , Fenilanina Amoníaco-Liasa/metabolismo , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Plantones/microbiología , Simbiosis/genética , Triticum/genética , Triticum/microbiologíaRESUMEN
Treatment of Linum album cell cultures with 10 µM salicylic acid (SA) for 3 days improved podophyllotoxin (PTOX) production up to 333 µg/g dry weight (DW): over three times that of the control cultures. qPCR analyses showed that in SA-treated cells, the expression of the genes coding for phenylalanine ammonia-lyase (PAL), cinnamoyl-CoA reductase (CCR) and cinnamyl-alcohol dehydrogenase (CAD), all involved in the first steps of PTOX biosynthesis, also increased reaching a peak 8-12 h after the treatment. Expression of the pinoresinol-lariciresinol reductase gene (PLR), which is involved in one of the last biosynthetic steps, was not affected by SA. The selective action of SA on these genes can be applied to control the biotechnological production of this anticancer agent.
Asunto(s)
Vías Biosintéticas , Lino/efectos de los fármacos , Lino/metabolismo , Expresión Génica/efectos de los fármacos , Proteínas de Plantas/biosíntesis , Podofilotoxina/biosíntesis , Ácido Salicílico/metabolismo , Oxidorreductasas de Alcohol/biosíntesis , Aldehído Oxidorreductasas/biosíntesis , Técnicas de Cultivo de Célula , Perfilación de la Expresión Génica , Oxidorreductasas/biosíntesis , Fenilanina Amoníaco-Liasa/biosíntesisRESUMEN
The cDNAs (Espals) encoding phenylalanine ammonia-lyase (PAL) were cloned from Ephedra sinica by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers and by 5' and 3'-rapid amplification of cDNA ends (RACE). 2166 bp of the open reading frame (ORF) encoded 722 amino acids; sequence analyses of Espal clones suggested that at least four isoforms of EsPAL (EsPAL1, 2, 3, 4) existed, with nine amino acids substitution in their sequences. Phylogenetic analysis of EsPAL and PALs from other plant species revealed that EsPAL and Pinus PAL formed a gymnosperm-type PAL subfamily. The recombinant EsPAL1 to 4 functionally catalyzed a PAL reaction and their K(m), V(max), K(cat) and K(cat)/K(m) values did not show significant differences. Semi-quantitative RT-PCR analysis indicated that the expression of Espal genes in the roots was higher than in the plant's aerial parts. In addition, the activity of PAL in the roots was also higher than in the aerial parts. These results suggest that Espal genes are expressed in the whole plant but are dominant in the roots rather than in the aerial parts.
Asunto(s)
Ephedra/enzimología , Fenilanina Amoníaco-Liasa/genética , Secuencia de Aminoácidos , Southern Blotting , Cromatografía Líquida de Alta Presión , Cinamatos/química , Cinamatos/metabolismo , Clonación Molecular , ADN Complementario/biosíntesis , ADN Complementario/genética , ADN de Plantas/biosíntesis , ADN de Plantas/genética , ADN de Plantas/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Fenilanina Amoníaco-Liasa/biosíntesis , Fenilanina Amoníaco-Liasa/química , Filogenia , Hojas de la Planta/química , Raíces de Plantas/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Phenylalanine ammonia-lyase (PAL) is an important enzyme in both plant development and pathogen defense. In all plants it is encoded by a multi-gene family, ranging in copy number from four in Arabidopsis to a dozen or more copies in some higher plants. Many studies indicate that alternate genes are differentially regulated in response to environmental stimuli. In this study, Southern blot and dot blot analyses in tomato indicate a surprisingly large family of related sequences with approximately 26 copies in the diploid genome, some easily distinguished by restriction enzyme digestion. Analyses of a BAC genome library suggest that the genes are generally not clustered. A more detailed comparison of the gene sequences using PCR to isolate the individual copies and reverse transcription-PCR to study the transcripts that they encode indicates a significant diversity in the gene sequences themselves, but surprisingly only one mRNA transcript can be detected even when additional expression is induced by pathogen growth or wounding. Consistent with previous reports in other plants, a parallel study with a closely related plant, the potato, indicates a much broader utilization of the PAL genes, highlighting the unusual nature of this family in tomato and of the mechanism(s) that silences so many members. Plant transformation analyses further demonstrate the presence of very active silencing, suggesting aggressive competition between PAL gene duplication and copy inactivation during PAL gene evolution.
Asunto(s)
Evolución Molecular , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Familia de Multigenes/fisiología , Fenilanina Amoníaco-Liasa/biosíntesis , Proteínas de Plantas/biosíntesis , Solanum lycopersicum/enzimología , Arabidopsis/enzimología , Arabidopsis/genética , Cromosomas Artificiales Bacterianos/genética , Dosificación de Gen/fisiología , Genoma de Planta/fisiología , Biblioteca Genómica , Solanum lycopersicum/genética , Fenilanina Amoníaco-Liasa/genética , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Solanum tuberosum/enzimología , Solanum tuberosum/genética , Especificidad de la EspecieRESUMEN
Red light, acting via phytochrome, stimulates phenylalanine ammonia lyase (PAL) activity in cotyledons and hypocotyls of tomato seedlings. The time course of photoinduction of PAL activity has a peak level at 4 h after which activity declines significantly. In tomato seedlings PAL activity comprised of three isoforms and light stimulated activity of all three isoforms. A polyclonal antibody raised against PAL purified from tomato leaves recognized PAL protein belonging to PAL-II and PAL-III isoforms. The mode of increase in PAL activity was investigated by immunochemical techniques. The photostimulated increase in PAL activity appeared to be dependent on de novo synthesis of protein and nucleic acid. However, inhibition of protein phosphatase activity blocked increase in PAL activity without affecting the increase in PAL protein levels. The results indicate that in addition to de novo synthesis, the photostimulation of PAL activity likely requires dephosphorylation by a type 2C protein phosphatase.
Asunto(s)
Cotiledón/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Hipocótilo/enzimología , Luz , Fenilanina Amoníaco-Liasa/biosíntesis , Solanum lycopersicum/enzimología , Isoenzimas/biosíntesisRESUMEN
To construct a safer and more efficient gene engineering Lactococcus Lactis for expressing phenylalaine ammonia lyase (PAL) which will be benefit for PKU therapy, pal cDNA of Parsly and synthesized sequence based on Lactococcus Lactis bias codons were recombined into two Lactococcus Lactis NICE systems. The activities of the expressed PAL were detected, and the effect of Lactococcus Lactis bias codons on the expression of exterior protein was analyzed. The results showed that the expression level of PAL was increased by using Lactococcus Lactis bias codons in both Lactococcus Lactis NICE systems. Through which several safer andmore efficient strains of the gene engineering Lactococcus Lactis were obtained.
Asunto(s)
Codón/genética , Lactococcus lactis/metabolismo , Fenilanina Amoníaco-Liasa/biosíntesis , Proteínas Recombinantes/biosíntesis , Clonación Molecular , Vectores Genéticos/genética , Lactococcus lactis/genética , Fenilanina Amoníaco-Liasa/genética , Proteínas Recombinantes/metabolismo , Transformación BacterianaRESUMEN
The accumulation of podophyllotoxin (PTOX) and 6-methoxypodophyllotoxin (6MPTOX) was enhanced about twofold in the suspension culture of Linum album line 2-5 aH following the addition of methyl jasmonate (MeJas) to the cultivation medium, reaching 7.69+/-1.45 mg/g dry weight and 1.11+/-0.09 mg/g dry weight, respectively. There was no increase in 6MPTOX accumulation following the addition of MeJas to suspension cells of L. album line X4SF, whereas PTOX accumulation was enhanced about tenfold to 0.49+/-0.10 mg/g dry weight. Phenylalanine ammonia-lyase activity increased immediately after the addition of MeJas to a cell suspension culture of line X4SF, reaching a maximum between 4 h and 1 day after elicitation, while cinnamyl alcohol dehydrogenase activity and the lignin content of the cells were not affected.
Asunto(s)
Acetatos/farmacología , Ciclopentanos/farmacología , Lino/metabolismo , Lignanos/biosíntesis , Podofilotoxina/biosíntesis , Oxidorreductasas de Alcohol/biosíntesis , Técnicas de Cultivo de Célula , Lino/efectos de los fármacos , Oxilipinas , Fenilanina Amoníaco-Liasa/biosíntesisRESUMEN
A Pseudomonas putida S12 strain was constructed that efficiently produced the fine chemical cinnamic acid from glucose or glycerol via the central metabolite phenylalanine. The gene encoding phenylalanine ammonia lyase from the yeast Rhodosporidium toruloides was introduced. Phenylalanine availability was the main bottleneck in cinnamic acid production, which could not be overcome by the overexpressing enzymes of the phenylalanine biosynthesis pathway. A successful approach in abolishing this limitation was the generation of a bank of random mutants and selection on the toxic phenylalanine anti-metabolite m-fluoro-phenylalanine. Following high-throughput screening, a mutant strain was obtained that, under optimised culture conditions, accumulated over 5 mM of cinnamic acid with a yield (Cmol%) of 6.7%.
Asunto(s)
Cinamatos/metabolismo , Glucosa/metabolismo , Fenilanina Amoníaco-Liasa/metabolismo , Pseudomonas putida/enzimología , Pseudomonas putida/genética , 3-Desoxi-7-Fosfoheptulonato Sintasa/metabolismo , Catálisis , Cinamatos/síntesis química , Cinamatos/química , Tolerancia a Medicamentos , Fermentación , Ingeniería Genética , Vectores Genéticos , Mutación , Fenilalanina/análogos & derivados , Fenilalanina/antagonistas & inhibidores , Fenilalanina/metabolismo , Fenilanina Amoníaco-Liasa/biosíntesis , Fenilanina Amoníaco-Liasa/genética , Prefenato Deshidratasa/metabolismo , Pseudomonas putida/crecimiento & desarrollo , Solventes/farmacologíaRESUMEN
We prepared a series of cryptogein mutants, an elicitor from Phytophthora cryptogea, with altered abilities to bind sterols and fatty acids. The induction of the early events, i.e., synthesis of active oxygen species and pH changes, in suspension tobacco cells by these mutated proteins was proportional to their ability to bind sterols but not fatty acids. Although the cryptogein-sterol complex was suggested to be a form triggering a defense reaction in tobacco, some proteins unable to bind sterols induced the synthesis of active oxygen species and pH changes. The modeling experiments showed that conformational changes after the introduction of bulky residues into the omega loop of cryptogein resemble those induced by sterol binding. These changes may be necessary for the ability to trigger the early events by elicitins. However, the ability to stimulate necrosis in suspension tobacco cells and the expression of defense proteins in tobacco plants were linked neither to the lipid binding capacity nor to the capacity to provoke the early events. On the basis of these experiments and previous results, we propose that elicitins could stimulate two signal pathways. The first one induces necroses and the expression of pathogen-related proteins, includes tyrosine protein kinases and mitogen-activated protein kinases, and depends on the overall structure and charge distribution. The second type of interaction is mediated by phospholipase C and protein kinase C. It triggers the synthesis of active oxygen species and pH changes. This interaction depends on the ability of elicitins to bind sterols.