RESUMEN

Recent achievements have been made in the metabolic engineering of plant secondary metabolism. Various pathways have been altered using genes encoding biosynthetic enzymes or genes encoding regulatory proteins. In addition, antisense genes have been used to block competitive pathways, thereby increasing the flux towards the desired secondary metabolites.

Asunto(s)

Enzimas/metabolismo , Ingeniería Genética/métodos , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Ingeniería de Proteínas/métodos

RESUMEN

Plant secondary metabolism is the source of many natural products with diverse applications, including pharmaceuticals, food colors, dyes and fragrances. Functions in plants include attraction of pollinating insects and protection against pests and pathogens. An important regulatory step in secondary metabolism is transcription of the biosynthetic genes. The aim of this chapter is to discuss results and opportunities concerning modification of secondary metabolism using transcriptional regulators. The transcriptional regulation of two well-studied secondary pathways, the phenylpropanoid pathway and its flavonoid branch, and the terpenoid indole alkaloid biosynthetic pathway, are reviewed. Some examples of successful engineering of these pathways via transcriptional regulators are discussed.

Asunto(s)

Regulación de la Expresión Génica de las Plantas , Genes Reguladores , Proteínas de Plantas , Plantas/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Ciclopentanos/química , Ciclopentanos/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Flavonoides/metabolismo , Genes de Plantas , Alcaloides Indólicos/metabolismo , Oxilipinas , Reguladores del Crecimiento de las Plantas/química , Reguladores del Crecimiento de las Plantas/metabolismo , Plantas/genética , Transducción de Señal , Terpenos/metabolismo , Técnicas del Sistema de Dos Híbridos

RESUMEN

Geraniol 10-hydroxylase (G10H) is a cytochrome P450 monooxygenase involved in the biosynthesis of iridoid monoterpenoids and several classes of monoterpenoid alkaloids found in a diverse range of plant species. Catharanthus roseus (Madagascar periwinkle) contains monoterpenoid indole alkaloids, several of which are pharmaceutically important. Vinblastine and vincristine, for example, find widespread use as anti-cancer drugs. G10H is thought to play a key regulatory role in terpenoid indole alkaloid biosynthesis. We purified G10H from C. roseus cells. Using degenerate PCR primers based on amino acid sequence information we cloned the corresponding cDNA. The encoded CYP76B6 protein has G10H activity when expressed in C. roseus and yeast cells. The stress hormone methyljasmonate strongly induced G10h gene expression coordinately with other terpenoid indole alkaloid biosynthesis genes in a C. roseus cell culture.

Asunto(s)

Alcaloides/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Magnoliopsida/enzimología , Magnoliopsida/genética , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Monoterpenos Acíclicos , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Clonación Molecular , Ciclopentanos/farmacología , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Cromatografía de Gases y Espectrometría de Masas , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Hidroxilación , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/aislamiento & purificación , Datos de Secuencia Molecular , Oxilipinas , Plantas Modificadas Genéticamente , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Especificidad por Sustrato , Terpenos/metabolismo , Levaduras

RESUMEN

A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for strictosidine synthase (STR) and strictosidine beta-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.

Asunto(s)

Frutas/metabolismo , Alcaloides Indólicos/metabolismo , Iridoides , Piranos/metabolismo , Saccharomyces cerevisiae/enzimología , Transgenes , Triptaminas/metabolismo , Biotecnología/métodos , Liasas de Carbono-Nitrógeno/genética , Liasas de Carbono-Nitrógeno/metabolismo , Medios de Cultivo , Frutas/química , Ingeniería Genética , Glucosidasas/genética , Glucosidasas/metabolismo , Glucósidos Iridoides , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo

RESUMEN

Jasmonic acid is an important plant stress signalling molecule. It induces the biosynthesis of defence proteins and protective secondary metabolites. In alkaloid metabolism, jasmonate acts by coordinate activation of the expression of multiple biosynthesis genes. In terpenoid indole alkaloid metabolism and primary precursor pathways, jasmonate induces gene expression and metabolism via ORCAs, which are members of the AP2/ERF-domain family of plant transcription factors. Other jasmonate-regulated (secondary) metabolic pathways might also be controlled by ORCA-like AP2/ERF-domain transcription factors. If so, such regulators could be used to improve plant fitness or metabolite productivity of plants or cell cultures.

Asunto(s)

Acetatos/metabolismo , Ciclopentanos/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Factores de Transcripción/metabolismo , Alcaloides de la Vinca/biosíntesis , Oxilipinas , Proteínas de Plantas/química , Proteínas de Plantas/fisiología , Transducción de Señal , Alcaloides de la Vinca/metabolismo

RESUMEN

The enzyme encoded by the strictosidine synthase (Str) gene catalyses a key step in the biosynthesis of therapeutically valuable terpenoid indole alkaloids. In Catharanthus roseus the Str gene was shown to be regulated by a wide variety of signals including auxin, methyl jasmonate and fungal elicitors in cell suspension cultures and by tissue-specific control in plant organs. The Str promoter contains a functional G-box (CACGTG) cis-regulatory sequence. In order to understand better the mechanisms involved in the regulation of Str gene expression, we isolated the C. roseus cDNAs encoding G-box binding factors Crgbf1 and Crgbf2. The binding specificity of their protein products CrGBF1 and CrGBF2 was analysed by competitive electrophoresis mobility and saturation binding assays. CrGBF1 had a high binding specificity for class I G-boxes including the Str G-box. CrGBF1 showed a lower affinity for class II G-boxes and for the G-box-like element (AACGTG) found in the tryptophan decarboxylase (Tdc) gene which encodes another enzyme involved in TIA biosynthesis. CrGBF2 showed a high affinity for all types of G-boxes tested and to a lesser extent for the Tdc G-box-like element. Transient bombardment experiments demonstrated that both CrGBF1 and CrGBF2 can act in vivo as transcriptional repressors of the Str promoter via direct interaction with the G-box. These data indicate that GBFs may play functional role in the regulation of expression of the terpenoid indole alkaloid biosynthetic gene Str.

Asunto(s)

Liasas de Carbono-Nitrógeno/genética , Proteínas de Unión al ADN/genética , Plantas/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Unión Competitiva , Northern Blotting , Southern Blotting , ADN/genética , ADN/metabolismo , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN de Plantas/genética , Proteínas de Unión al ADN/metabolismo , Factores de Unión a la G-Box , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Células Vegetales , Regiones Promotoras Genéticas , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución Tisular , Factores de Transcripción/metabolismo

RESUMEN

The AP2/ERF-domain transcription factor ORCA3 is a master regulator of primary and secondary metabolism in Catharanthus roseus (periwinkle). Here we demonstrate that ORCA3 specifically binds to and activates gene expression via a previously characterized jasmonate- and elicitor-responsive element (JERE) in the promoter of the terpenoid indole alkaloid biosynthetic gene Strictosidine synthase (Str). Functional characterization of different domains in the ORCA3 protein in yeast and plant cells revealed the presence of an N-terminal acidic activation domain and a serine-rich C-terminal domain with a negative regulatory function. Orca3 mRNA accumulation was rapidly induced by the plant stress hormone methyljasmonate with biphasic kinetics. A precursor and an intermediate of the jasmonate biosynthetic pathway also induced Orca3 gene expression, further substantiating the role for ORCA3 in jasmonate signaling. The protein synthesis inhibitor cycloheximide did not inhibit jasmonate-responsive expression of Orca3, nor of its target genes Str and Tryptophan decarboxylase (Tdc). In conclusion, ORCA3 regulates jasmonate-responsive expression of the Str gene via direct interaction with the JERE. The activating activities of ORCA proteins do not seem to depend on jasmonate-induced de novo protein synthesis, but presumably occur via modification of pre-existing ORCA protein.

Asunto(s)

Asteraceae/genética , Ciclopentanos/farmacología , Regulación de la Expresión Génica de las Plantas , Regiones Promotoras Genéticas/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Asteraceae/metabolismo , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes Reporteros , Datos de Secuencia Molecular , Oxilipinas , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ARN Mensajero/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Activación Transcripcional

RESUMEN

T-DNA activation tagging is a method used to generate dominant mutations in plants or plant cells by the insertion of a T-DNA which carries constitutive enhancer elements that can cause transcriptional activation of flanking plant genes. We applied this approach to the species Catharanthus roseus (L.) G. Don (Madagascar periwinkle), in an attempt to isolate regulators of genes that are involved in the biosynthesis of secondary metabolites of the terpenoid indole alkaloid (TIA) class. Several TIAs have pharmaceutically interesting activities, including the anti-tumour agents vincristine and vinblastine. The use of suspension-cultured cells enabled us to screen in a relatively easy way hundreds of thousands of T-DNA-tagged cells for resistance to a toxic substrate of one of the TIA biosynthetic enzymes: tryptophan decarboxylase. This screening yielded several interesting tagged cell lines. Further characterisation of one of the tagged cell lines led to the isolation of Orca3, a gene encoding an AP2/ERF-domain transcription factor that acts as a master regulator of primary and secondary metabolism. The T-DNA activation tagging results described in detail in this paper illustrate the usefulness of this approach to isolate regulators of a complex metabolic pathway from a genetically non-tractable plant species.

Asunto(s)

Catharanthus/genética , ADN Bacteriano , Genes de Plantas , Plantas/genética , Descarboxilasas de Aminoácido-L-Aromático/genética , Northern Blotting , Southern Blotting , Catharanthus/clasificación , Células Cultivadas , ADN de Cadena Simple , Genes Dominantes , Mutación , Plásmidos/metabolismo

RESUMEN

Plant secondary metabolism is very important for traits such as flower color, flavor of food, and resistance against pests and diseases. Moreover, it is the source of many fine chemicals such as drugs, dyes, flavors, and fragrances. It is thus of interest to be able to engineer the secondary metabolite production of the plant cell factory, e.g. to produce more of a fine chemical, to produce less of a toxic compound, or even to make new compounds, Engineering of plant secondary metabolism is feasible nowadays, but it requires knowledge of the biosynthetic pathways involved. To increase secondary metabolite production different strategies can be followed, such as overcoming rate limiting steps, reducing flux through competitive pathways, reducing catabolism and overexpression of regulatory genes. For this purpose genes of plant origin can be overexpressed, but also microbial genes have been used successfully. Overexpression of plant genes in microorganisms is another approach, which might be of interest for bioconversion of readily available precursors into valuable fine chemicals. Several examples will be given to illustrate these various approaches. The constraints of metabolic engineering of the plant cell factory will also be discussed. Our limited knowledge of secondary metabolite pathways and the genes involved is one of the main bottlenecks.

Asunto(s)

Ingeniería Genética/métodos , Plantas/química , Plantas/genética , Reactores Biológicos , Regulación de la Expresión Génica , Genes de Plantas/genética , Células Vegetales , Transformación Genética , Levaduras/genética

RESUMEN

This paper describes a so-called ternary transformation system for plant cells. We demonstrate that Agrobacterium tumefaciens strain LBA4404 supplemented with a constitutive virG mutant gene (virGN54D) on a compatible plasmid is capable of very efficient T-DNA transfer to a diverse range of plant species. For the plant species Catharanthus roseus it is shown that increased T-DNA transfer results in increased stable transformation frequencies. Analysis of stably transformed C. roseus cell lines showed that, although the T-DNA transfer frequency is greatly enhanced by addition of virGN54D, only one or a few T-DNA copies are stably integrated into the plant genome. Thus, high transformation frequencies of different plant species can be achieved by introduction of a ternary plasmid carrying a constitutive virG mutant into existing A. tumefaciens strains in combination with standard binary vectors.

Asunto(s)

Transformación Genética , Acetofenonas/farmacología , Agrobacterium tumefaciens/citología , Agrobacterium tumefaciens/efectos de los fármacos , Agrobacterium tumefaciens/genética , Arabidopsis/citología , Arabidopsis/genética , Proteínas Bacterianas , Técnicas de Cocultivo , ADN Bacteriano/efectos de los fármacos , ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , Mutación , Células Vegetales , Plantas/efectos de los fármacos , Plantas/genética , Plásmidos/genética , Factores de Transcripción/genética

RESUMEN

Biosynthesis of many classes of secondary metabolites in plants is induced by the stress hormone jasmonate. The gene for ORCA3, a jasmonate-responsive APETALA2 (AP2)-domain transcription factor from Catharanthus roseus, was isolated by transferred DNA activation tagging. Orca3 overexpression resulted in enhanced expression of several metabolite biosynthetic genes and, consequently, in increased accumulation of terpenoid indole alkaloids. Regulation of metabolite biosynthetic genes by jasmonate-responsive AP2-domain transcription factors may link plant stress responses to changes in metabolism.

Asunto(s)

Regulación de la Expresión Génica de las Plantas , Magnoliopsida/genética , Proteínas de Plantas/genética , Factores de Transcripción/genética , Acetatos/farmacología , Línea Celular , Ciclopentanos/farmacología , ADN Bacteriano , Proteínas de Homeodominio/química , Datos de Secuencia Molecular , Proteínas Nucleares/química , Oxilipinas , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/química , Proteínas de Plantas/fisiología , Factores de Transcripción/química , Factores de Transcripción/fisiología , Alcaloides de la Vinca/biosíntesis , Alcaloides de la Vinca/metabolismo

RESUMEN

Strictosidine beta-D-glucosidase (SGD) is an enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIAs) by converting strictosidine to cathenamine. The biosynthetic pathway toward strictosidine is thought to be similar in all TIA-producing plants. Somewhere downstream of strictosidine formation, however, the biosynthesis diverges to give rise to the different TIAs found. SGD may play a role in creating this biosynthetic diversity. We have studied SGD at both the molecular and enzymatic levels. Based on the homology between different plant beta-glucosidases, degenerate polymerase chain reaction primers were designed and used to isolate a cDNA clone from a Catharanthus roseus cDNA library. A full-length clone gave rise to SGD activity when expressed in Saccharomyces cerevisiae. SGD shows approximately 60% homology at the amino acid level to other beta-glucosidases from plants and is encoded by a single-copy gene. Sgd expression is induced by methyl jasmonate with kinetics similar to those of two other genes acting prior to Sgd in TIA biosynthesis. These results show that coordinate induction of the biosynthetic genes forms at least part of the mechanism for the methyl jasmonate-induced increase in TIA production. Using a novel in vivo staining method, subcellular localization studies of SGD were performed. This showed that SGD is most likely associated with the endoplasmic reticulum, which is in accordance with the presence of a putative signal sequence, but in contrast to previous localization studies. This new insight in SGD localization has significant implications for our understanding of the complex intracellular trafficking of metabolic intermediates during TIA biosynthesis.

Asunto(s)

Alcaloides Indólicos , Proteínas de Plantas/genética , Plantas/enzimología , Alcaloides de la Vinca/metabolismo , beta-Glucosidasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Genes de Plantas , Datos de Secuencia Molecular , Proteínas de Plantas/análisis , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae , Alineación de Secuencia , Terpenos/metabolismo , beta-Glucosidasa/análisis , beta-Glucosidasa/metabolismo

RESUMEN

Plants respond to pathogen attack by induction of various defence responses, including the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the elicitor-induced expression of the terpenoid indole alkaloid biosynthetic gene Strictosidine synthase (Str) is mediated via the plant stress hormonejasmonate. In the promoters of several defence-related genes, cis-acting elements have been identified that are important for transcriptional regulation upon stress signals. Here we show that an upstream region in the Str promoter confers responsiveness to partially purified yeast elicitor and jasmonate. Yeast one-hybrid screening with this element as a bait identified a MYB-like protein, which shows high homology to parsley box P-binding factor-1 (PcBPF-1). In vitro analyses showed that the Str promoter fragment contained a novel binding site for BPF-1-like proteins with higher binding affinity than the previously described box P. CrBPF-1 mRNA accumulated rapidly in elicitor-treated C. roseus suspension cells, whereas no induction was observed with jasmonate. Inhibitor studies indicated that CrBPF-1 plays a role in an elicitor-responsive but jasmonate-independent signal transduction pathway, acting downstream of protein phosphorylation and calcium influx.

Asunto(s)

Liasas de Carbono-Nitrógeno/metabolismo , Ciclopentanos/farmacología , Proteínas de Unión al ADN/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Liasas de Carbono-Nitrógeno/genética , Huella de ADN , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN de Plantas/genética , ADN de Plantas/metabolismo , Proteínas de Unión al ADN/genética , Desoxirribonucleasas , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Datos de Secuencia Molecular , Oxilipinas , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transducción de Señal/efectos de los fármacos , Técnicas del Sistema de Dos Híbridos

RESUMEN

The tryptophan decarboxylase (Tdc) gene encodes a key enzyme in the biosynthesis of terpenoid indole alkaloids (TIAs) in Catharanthus roseus. TIAs absorb ultraviolet light (UV) and putative functions in plants include a role as UV protectants. In support of this possible function we demonstrate here that UV light induces accumulation of several TIAs as well as expression of the Tdc gene in C. roseus. In addition, in tobacco a Tdc-gusA construct was found to be specifically induced by UV-B light. Lack of induction by UV-A or other wavelengths of light indicate that Tdc expression is regulated by a specific UV-B receptor and corresponding signal transduction pathway. To identify UV-responsive Tdc promoter elements, a loss-of-function analysis was performed, in which deletion derivatives were fused to the gusA reporter gene and analysed in transgenic tobacco plants. Truncation of the Tdc promoter from -1818 (relative to the start of transcription) to -160 reduced expression levels two-fold without affecting the qualitative UV response. Deletion to -37 further reduced expression levels five-fold, but the delta37 promoter also remained UV-responsive. Subsequently, the -160 to -37 region was further studied by gain-of-function experiments, in which the transcriptional activities of tetramerized subfragments fused to truncated promoters were analysed. Combination of the data identified several functional regions in the -160 to +198 promoter. The - 160 to -99 region acts as the main transcriptional enhancer. UV-responsive elements appeared to be redundant in the -160 Tdc promoter and to reside between -99 and -37 and between -37 and + 198.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático/genética , Plantas/efectos de la radiación , Regiones Promotoras Genéticas/genética , Elementos de Respuesta/efectos de la radiación , Rayos Ultravioleta , Alcaloides/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Liasas de Carbono-Nitrógeno/genética , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Glucuronidasa/genética , Glucuronidasa/metabolismo , Datos de Secuencia Molecular , Plantas/enzimología , Plantas/genética , Plantas Modificadas Genéticamente , Plantas Tóxicas , Regiones Promotoras Genéticas/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta/genética , Eliminación de Secuencia , Transducción de Señal/efectos de la radiación , Nicotiana/genética , Nicotiana/efectos de la radiación

RESUMEN

Jasmonate (JA) is an important plant stress hormone that induces various plant defense responses, including the biosynthesis of protective secondary metabolites. The induction of the secondary metabolite biosynthetic gene Strictosidine synthase (Str) in Catharanthus roseus (periwinkle) cells by elicitor requires JA as a second messenger. A 42 bp region in the Str promoter is both necessary and sufficient for JA- and elicitor-responsive expression. This region is unlike other previously identified JA-responsive regions, and contains a GCC-box-like element. Yeast one-hybrid screening identified cDNAs encoding two AP2-domain proteins. These octadecanoid-derivative responsive Catharanthus AP2-domain (ORCA) proteins bind in a sequence-specific manner the JA- and elicitor-responsive element. ORCA2 trans-activates the Str promoter and its expression is rapidly inducible with JA and elicitor, whereas Orca1 is expressed constitutively. The results indicate that a GCC-box-like element and ORCA2 play key roles in JA- and elicitor-responsive expression of the terpenoid indole alkaloid biosynthetic gene Str.

Asunto(s)

Acetatos/farmacología , Liasas de Carbono-Nitrógeno/genética , Ciclopentanos/farmacología , Regiones Promotoras Genéticas , Elementos de Respuesta , Transactivadores/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario , ADN de Plantas , Datos de Secuencia Molecular , Oxilipinas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , ARN Mensajero , Transactivadores/genética

RESUMEN

Plant secondary metabolites of the terpenoid indole alkaloid (TIA) class comprise several compounds with pharmaceutical applications. A key step in the TIA biosynthetic pathway is catalysed by the enzyme tryptophan decarboxylase (TDC), which channels the primary metabolite tryptophan into TIA metabolism. In Catharanthus roseus (Madagascar periwinkle), the Tdc gene is expressed throughout plant development. Moreover, Tdc gene expression is induced by external stress signals, such as fungal elicitor and UV light. In a previous study of Tdc promoter architecture in transgenic tobacco it was shown that the -538 to -112 region is a quantitative determinant for the expression level in different plant organs. Within this sequence one particular region (-160 to -99) was identified as the major contributor to basal expression and another region (-99 to -37) was shown to be required for induction by fungal elicitor. Here, the in vitro binding of nuclear factors to the -572 to -37 region is described. In extracts from tobacco and C. roseus, two binding activities were detected that could be identified as the previously described nuclear factors GT-1 and 3AF1, based on their mobility and binding characteristics. Both factors appeared to interact with multiple regions in the Tdc promoter. Mutagenesis of GT-1 binding sites in the Tdc promoter did not affect the basal or elicitor-induced expression levels. However, induction of the Tdc promoter constructs by UV light was significantly lower, thereby demonstrating a functional role for GT-1 in the induction of Tdc expression by UV light.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Proteínas Nucleares/metabolismo , Proteínas de Plantas/metabolismo , Plantas/genética , Regiones Promotoras Genéticas , Rayos Ultravioleta , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Secuencia de Bases , Sitios de Unión , Núcleo Celular/metabolismo , Células Cultivadas , Madagascar , Datos de Secuencia Molecular , Plantas/efectos de la radiación , Plantas Modificadas Genéticamente , Plantas Tóxicas , TATA Box , Nicotiana , Factores de Transcripción/metabolismo , Dedos de Zinc

RESUMEN

The enzyme encoded by the strictosidine synthase (Str) gene from Catharanthus roseus catalyses a key step in the biosynthesis of the pharmaceutically important terpenoid indole alkaloids. Str cDNA and genomic clones have already been isolated, allowing us to study the regulation of Str gene expression. Here we focus on the role of a putative cis-acting element, CACGTG, in the Str promoter. This sequence is known as a G-box, and functions as a transcription-regulating sequence in a number of other promoters. By means of electrophoretic mobility shift assays it was demonstrated that the Str G-box is capable of interacting with nuclear factors in tobacco and with the cloned tobacco G-box-binding factor TAF-1. Disruption of the Str G-box sequence by two single-nucleotide mutations prevented binding of factors, thereby demonstrating the specificity of the observed interactions. Functional analysis in transgenic tobacco plants demonstrated that these mutations also reduced the transcriptional activity of constructs containing tetramers of the Str G-box sequence. Expression directed by a tetramer of the Str G-box fused to a truncated promoter containing only a TATA box was confined to seeds and was found to increase during seed maturation. Thus, the Str G-box tetramer is able to direct seed-specific expression independently of other regulatory sequences. G-box-directed expression in leaves required the presence of an enhancer region from the cauliflower mosaic virus (CaMV) 35S promoter. The results indicate that the G-box needs to interact with other elements to drive expression in leaf, and that it can by itself confer seed-specific expression as a multimer. The fact that only some of the G-boxes found in different promoters serve as seed-specific elements indicates that sequences flanking the G-box determine the transcriptional activity in different tissues. Based on sequence comparisons we propose that the nucleotides at positions -4, -3, -2 and/or +4 are important in determining seed-specific expression.

Asunto(s)

Liasas de Carbono-Nitrógeno/genética , Plantas/enzimología , Plantas/genética , Regiones Promotoras Genéticas , Secuencia de Bases , Liasas de Carbono-Nitrógeno/biosíntesis , Clonación Molecular , ADN Complementario , Glucuronidasa/genética , Hojas de la Planta , Plantas Modificadas Genéticamente , Plantas Tóxicas , Proteínas Recombinantes de Fusión/biosíntesis , Mapeo Restrictivo , Semillas/enzimología , TATA Box , Nicotiana/genética

RESUMEN

Strictosidine synthase (STR) is a key enzyme in the biosynthesis of terpenoid indole alkaloids. This class of secondary metabolites harbours several pharmaceutically important compounds used, among other applications, in cancer treatment. Terpenoid indole alkaloid biosynthesis and expression of biosynthetic genes including Str1 is induced by fungal elicitors. To identify elicitor-responsive regulatory promoter elements and trans-acting factors, the single-copy Str1 gene was isolated from the subtropical plant species Catharanthus roseus (Madagascar periwinkle). Str1 upstream sequences conferred elicitor-responsive expression to the beta-glucuronidase (gusA) reporter gene in transgenic tobacco plants. Main enhancer sequences within the Str1 promoter region studied were shown to be located between -339 and -145. This region and two other regions of the promoter bound the tobacco nuclear protein factor GT-1. A G-box located around position -105 bound nuclear and cloned G-box-binding factors (GBFs). A mutation that knocked out GBF binding had no measurable effect on expression, which indicates that the G-box is not essential for the elicitor responsiveness of the Str1 promoter. No obvious homologies with promoter elements identified in other elicitor-responsive genes were observed, suggesting that the Str1 gene may depend on novel regulatory mechanisms.

Asunto(s)

Liasas de Carbono-Nitrógeno/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Magnoliopsida/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Secuencia de Bases , Extractos Celulares/farmacología , Clonación Molecular , Huella de ADN , Sondas de ADN , Elementos de Facilitación Genéticos/genética , Factores de Unión a la G-Box , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas/genética , Genes Reporteros , Magnoliopsida/enzimología , Datos de Secuencia Molecular , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/genética , Plantas Modificadas Genéticamente , Plantas Tóxicas , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Respuesta/genética , Eliminación de Secuencia , Nicotiana/efectos de los fármacos , Nicotiana/genética , Transactivadores/metabolismo

RESUMEN

Two key genes in terpenoid indole alkaloid biosynthesis, Tdc and Str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-cultured Catharanthus roseus cells. We have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract. In addition to activating Tdc and Str gene expression, the elicitor also induced the biosynthesis of jasmonic acid. The jasmonate precursor alpha-linolenic acid or methyl jasmonate (MeJA) itself induced Tdc and Str gene expression when added exogenously. Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both the elicitor-induced formation of jasmonic acid and the activation of terpenoid indole alkaloid biosynthetic genes. The protein kinase inhibitor K-252a abolished both elicitor-induced jasmonate biosynthesis and MeJA-induced Tdc and Str gene expression. Analysis of the expression of Str promoter/gusA fusions in transgenic C. roseus cells showed that the elicitor and MeJA act at the transcriptional level. These results demonstrate that the jasmonate biosynthetic pathway is an integral part of the elicitor-triggered signal transduction pathway that results in the coordinate expression of the Tdc and Str genes and that protein kinases act both upstream and downstream of jasmonates.

RESUMEN

The tryptophan decarboxylase (Tdc) gene from Catharanthus roseus (Madagascar periwinkle) encodes a key enzyme in biosynthesis of terpenoid indole alkaloids. The expression of the Tdc gene is transcriptionally induced by fungal elicitors. Tdc upstream sequences from -1818 to +198 relative to the transcriptional start site were functionally analysed to identify cis-acting elements that determine basal expression or respond to elicitor. In a loss-of-function analysis promoter derivatives with 5' or internal deletions fused to the gusA reporter gene were analysed in transgenic tobacco plants. Whereas promoter activity dropped considerably following deletion down to -160, this short promoter derivative was still elicitor-responsive. Subsequently, the -160 to -37 region was further studied by gain-of-function experiments, in which subfragments were tested as tetramers cloned on two different truncated promoters. Combination of the data resulted in the identification of three functional regions in the -160 promoter. The region between -160 to -99 was shown to act as the main transcriptional enhancer. Two separable elicitor-responsive elements were found to reside between -99 and -87 and between -87 and -37. These two elements are not redundant in the Tdc promoter, since their combination gave a distinct elicitor response.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático/genética , Plantas/enzimología , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Vectores Genéticos , Datos de Secuencia Molecular , Plantas/genética , Plantas Modificadas Genéticamente , Plantas Tóxicas , Nicotiana/genética , Transfección