RESUMEN
Intra-nucleosomal cleavage of DNA into fragments of about 200 bp was demonstrated to occur in developing anthers, in which microspores had developed into the mid-late to late uni-nucleate stage in situ, i.e. at the verge of mitosis. The same was observed, but to a much larger extent, if these anthers were pre-treated by a hyper-osmotic shock. Pretreatment of anthers before the actual culture of microspores was required for optimal androgenesis of microspores. The use of the TUNEL reaction, which specifically labels 3' ends of DNA breaks, after intra-nucleosomal cleavage of DNA, revealed that DNA fragmentation mainly occurred in the loculus wall cells, tapetum cells and filament cells. TUNEL staining was absent or infrequently observed in the microspores of developing anthers in situ. Electron microscopy studies showed condensed chromatin in nuclei of loculus wall cells in the developing anthers. These observations at the chromatin and DNA level are known characteristics of programmed cell death, also known as apoptosis. Features of apoptosis were infrequently found in microspores from freshly isolated mature anthers. However, most tapetum cells had disappeared in these anthers and the remaining cell structures showed loss of cellular content. The viability of microspores in pre-treated anthers was comparable to those in freshly isolated anthers and almost four times higher than in anthers from control experiments. This observation was correlated with three to four times less microspores showing TUNEL staining and a two times higher level of ABA in the anther plus medium samples than in controls. Addition of ABA to the controls enhanced the viability and lowered the occurrence of apoptosis linked characteristics in the microspores. These data suggest that pre-treatment is effective in stimulating androgenesis because it leads to an increase in ABA levels which protects microspores from dying by apoptosis.
Asunto(s)
Ácido Abscísico/fisiología , Apoptosis , Hordeum/fisiología , Polen/embriología , Ácido Abscísico/metabolismo , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , ADN de Plantas/efectos de los fármacos , ADN de Plantas/genética , Diuréticos Osmóticos/farmacología , Fluoresceína , Haploidia , Hordeum/genética , Hordeum/ultraestructura , Etiquetado Corte-Fin in Situ , Manitol/farmacología , Microscopía Electrónica , Microscopía Fluorescente , Polen/efectos de los fármacos , Polen/crecimiento & desarrollo , ARN de Planta/efectos de los fármacos , ARN de Planta/genéticaRESUMEN
This paper describes the characterization of Oshox1, a cDNA clone from rice encoding a member of the homeodomain-leucine zipper (HD-Zip) class of putative transcription factors. Oshox1 maps to chromosome 10 and belongs to a family of related rice genes. Two-hybrid assays showed that Oshox1 protein can homodimerize, but can also form heterodimers with an Arabidopsis HD-Zip protein. This suggests that protein-protein interactions may also occur between different HD-Zip proteins in rice, which would provide enormous versatility for generating specific gene-control mechanisms. Oshox1 mRNA could be detected in various rice tissues at different developmental stages, with highest levels in embryos, shoots of seedlings, and leaves of mature plants. Transgenic expression of Oshox1 in Arabidopsis retarded growth and affected leaf size and shape, indicative of a role as developmental regulator. In vitro and in vivo DNA-binding studies revealed that Oshox1 interacts with the pseudopalindromic sequence CAAT(C/G)ATTG, confirming that the protein represents a transcription factor. Oshox1 was found to repress reporter gene activity in rice suspension cells, most likely by a mechanism of active transcriptional repression. Repression was strictly dependent on the presence of upstream Oshox1 binding sites in the reporter gene constructs and a function of the N-terminal region of Oshox1, preceding the homeodomain.
Asunto(s)
Proteínas de Homeodominio/genética , Leucina Zippers , Oryza/genética , Proteínas de Plantas , Factores de Transcripción/genética , Transcripción Genética/fisiología , Secuencia de Aminoácidos , Células Cultivadas , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/genética , Dimerización , Dosificación de Gen , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/metabolismo , Datos de Secuencia Molecular , Filogenia , Plantas Modificadas Genéticamente , ARN Mensajero/análisis , ARN de Planta/análisis , Proteínas Recombinantes de Fusión , Factores de Transcripción/metabolismoRESUMEN
During germination of barley grains, DNA fragmentation was observed in the aleurone. The appearance of DNA fragmentation in the aleurone layer, observed by TUNEL staining in aleurone sections, started near the embryo and extended to the aleurone cells far from the embryo in a time dependent manner. The same spatial temporal activities of hydrolytic enzymes such as alpha-amylase were observed in aleurone. DNA fragmentation could also be seen in vitro under osmotic stress, in isolated aleurone. During aleurone protoplast isolation, a very enhanced and strong DNA fragmentation occurred which was not seen in protoplast preparations of tobacco leaves. ABA was found to inhibit DNA fragmentation occurring in barley aleurone under osmotic stress condition and during protoplast isolation, while the plant growth regulator gibberellic acid counteracted the effect of ABA. Addition of auxin or cytokinin had no significant effect on DNA fragmentation in these cells. To study the role of phosphorylation in ABA signal transduction leading to control of DNA fragmentation (apoptosis), the effects of the phosphatase inhibitor okadaic acid and of phenylarisine oxide on apoptosis were studied. We hypothesize that the regulation of DNA fragmentation in aleurone plays a very important role in spatial and temporal control of aleurone activities during germination. The possible signal transduction pathway of ABA leading to the regulation of DNA fragmentation is discussed.
Asunto(s)
Ácido Abscísico/farmacología , Apoptosis , Hordeum/citología , Semillas/citología , Apoptosis/efectos de los fármacos , Arsenicales/farmacología , Fragmentación del ADN , Inhibidores Enzimáticos/farmacología , Germinación , Giberelinas/farmacología , Hordeum/efectos de los fármacos , Hordeum/embriología , Ácido Ocadaico/farmacología , Fosforilación , Protoplastos/efectos de los fármacos , Protoplastos/metabolismo , Semillas/efectos de los fármacos , Semillas/crecimiento & desarrollo , Transducción de SeñalRESUMEN
The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) catalyses a key step in the biosynthesis of terpenoid indole alkaloids in C. roseus by converting tryptophan into tryptamine. Hardly any tdc mRNA could be detected in hormone-independent callus and cell suspension cultures transformed by the oncogenic T-DNA of Agrobacterium tumefaciens. Supply of tryptamine may therefore represent a limiting factor in the biosynthesis of alkaloids by such cultures. To investigate this possibility, chimaeric gene constructs, in which a tdc cDNA is linked in the sense or antisense orientation to the cauliflower mosaic virus 35S promoter and terminator, were introduced in C. roseus cells by infecting seedlings with an oncogenic A. tumefaciens strain. In the resulting crown gall tumour calluses harbouring the tdc sense construct, an increased TDC protein level, TDC activity and tryptamine content but no significant increase in terpenoid indole alkaloid production were observed compared to empty-vector-transformed tumour calluses. In tumour calluses containing the tdc antisense construct, decreased levels of TDC activity were measured. Factors which might be responsible for the lack in increased terpenoid indole alkaloid production in the tdc cDNA overexpressing crown gall calluses are discussed.
Asunto(s)
Alcaloides/metabolismo , Descarboxilasas de Aminoácido-L-Aromático/genética , Indoles/metabolismo , Tumores de Planta/química , Plantas/enzimología , Alcaloides/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Regulación de la Expresión Génica de las Plantas , Vectores Genéticos/química , Indoles/química , Datos de Secuencia Molecular , Plantas/genética , Plantas/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Conejos , Regiones Terminadoras Genéticas , Terpenos/química , Terpenos/metabolismo , Transcripción Genética , Triptaminas/metabolismoRESUMEN
The enzyme tryptophan decarboxylase (TDC; EC 4.1.1.28) converts tryptophan into tryptamine. In Catharanthus roseus and other plants capable of producing terpenoid indole alkaloids (TIAs) TDC links primary metabolism to the secondary metabolic pathway involved in the biosynthesis of these compounds. The accumulation of tdc mRNA in C. roseus cells is developmentally regulated and transcriptionally influenced by elicitors (induction) and auxins (repression). Here we report that TDC is encoded by a single copy gene in the C. roseus genome. No introns were observed upon isolation and sequencing of this gene. To study gene expression controlled by the tdc promoter, a 2 kb promoter fragment and a number of 5' deleted promoter derivatives were joined in translational fusion to a beta-D-glucuronidase reporter gene (gusA). Expression of the chimaeric constructs was monitored in stably transformed tobacco plants and in transiently transfected tobacco protoplasts. Histochemical and fluorimetric analysis of transgenic plants revealed that 1938 bp of the tdc promoter (with respect to the translational start codon) give rise to GUS activity in roots, stems and leaves. No tissue or cell type specificity was noted. Promoter deletions up to nucleotide -398 directed lower levels of gusA expression but conferred the same pattern of staining for GUS activity as the -1938 construct. Further deletion of the tdc promoter up to nucleotide -232 resulted in drastically reduced GUS activity levels and loss of GUS staining in all parts of the transgenic plants. In contrast to stable transformation, the -232 tdc-gusA construct gave rise to GUS activity levels comparable to those of the -398 construct in an assay system for transient expression in protoplasts.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Descarboxilasas de Aminoácido-L-Aromático/genética , Genes de Plantas , Nicotiana/genética , Plantas Medicinales/enzimología , Plantas Medicinales/genética , Plantas Tóxicas , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Expresión Génica , Genes Reporteros , Glucuronidasa/genética , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , ARN Mensajero/genética , Transfección , Transformación GenéticaRESUMEN
A tobacco cDNA clone (pCNT1) was characterized that encodes an extensin apoprotein almost entirely composed of the repeats Ser-Pro4(-Lys2), Pro-Tyr2-Pro2-His and Thr-Pro-Val-Tyr-Lys. In healthy plants extensin transcripts are abundant in the roots, less prevalent in the stem and rare in the leaves. In leaves extensin mRNA is induced by wounding, ethylene or virus infection. Tobacco was transformed with pCNT1 cDNA coupled in sense or antisense orientation to the CaMV 35S promoter. Analysis of transgenic plants that over- or underexpressed pCNT1 mRNA demonstrated that the encoded protein constituted the majority of hydroxyproline-rich glycoproteins in roots, stems and leaves. The pCNT1-encoded protein contained at least 50% of total hydroxyproline present in these organs and was abundant in the soluble protein fraction of stems and roots as well as in the cell wall of stem vascular bundles. Analysis of transgenic plants expressing sense or antisense extensin gene constructs showed no correlation between total hydroxyproline concentration or soluble HRGP content and plant development.
Asunto(s)
Glicoproteínas/química , Nicotiana/genética , Proteínas de Plantas/química , Plantas Tóxicas , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , ADN , Epítopos/metabolismo , Regulación de la Expresión Génica , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Hidroxiprolina/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , ARN sin Sentido/biosíntesis , ARN Mensajero/biosíntesisRESUMEN
Transcriptional and translational fusions were made between the reading frame coding for beta-D-glucuronidase and sequences of either a constitutively expressed rice gene (GOS2) involved in initiation of translation or a light-inducible rice gene (GOS5). The transient expression of the fusions was studied via particle bombardment of seedling tissues of rice, perennial ryegrass and barley. Furthermore, the results of transient and stable expression were compared for cell suspensions of four rice varieties, one barley variety and one perennial ryegrass variety. The GOS2-gusA fusions were active in all three monocots studied. Best results were obtained for a construct having both a transcriptional and a translational fusion as well as intron and exon sequences (PORCEHyg). The level of GUS activity was in the range of activities as obtained by the 35S CaMV promoter transcriptionally fused to gusA. The gusA fusion with the light-inducible gene (GOS5) was active in green seedling tissues of all monocots studied. Also a weak expression compared to the GOS2 constructs was found in stably transformed rice callus. The gusA fusions with the mannopine synthase promoters 1' and 2' of the TR-DNA were transiently expressed at lower levels in cell suspensions than PORCEHyg. For stably transformed rice callus the expression of the GOS2-gusA fusion often decreased during prolonged subculture. This decrease in GUS activity and the various GUS-staining phenotypes of transgenic calli are explained by the presence of different cell types in the suspensions used and in the calli. It is presumed that the nature of the cells and their relative contribution in the calli change drastically upon further subculture.
Asunto(s)
Genes de Plantas , Hordeum/genética , Lolium/genética , Oryza/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Complejo de Proteína del Fotosistema I , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Secuencia de Bases , Técnicas de Cultivo , Regulación de la Expresión Génica , Glucuronidasa/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , Transformación GenéticaRESUMEN
The cell-type-specific expression of three rice genes, GOS2, GOS5 and GOS9, was studied by mRNA in situ hybridization. Previous northern blot analysis revealed that these genes were constitutive, green tissue-specific and root-specific, respectively. In this study, GOS2 transcripts were observed in all leaf cell types. In roots, a temporal and spatial expression pattern was noticed. Higher mRNA levels were observed in lateral roots, especially in parenchymal cells of the vascular cylinder. Expression of GOS5 was mainly found in chloroplast-containing cells. For GOS9, significant levels of signal were observed in root and leaf sections.
Asunto(s)
Regulación de la Expresión Génica , Genes de Plantas , Oryza/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Complejo de Proteína del Fotosistema I , Proteínas de Plantas/genética , Hibridación in Situ , Oryza/anatomía & histología , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/genética , Distribución Tisular , Transcripción GenéticaRESUMEN
Transcriptional and translational fusions were made between the reading frame coding for beta-D-glucuronidase and sequences of either a constitutively expressed rice gene (GOS2) involved in initiation of translation or a light-inducible rice gene (GOS5). The transient expression of the fusions was studied via particle bombardment of seedling tissues of rice, perennial ryegrass and barley. Furthermore, the results of transient and stable expression were compared for cell suspensions of four rice varieties, one barley variety and one perennial ryegrass variety. The GOS2-gusA fusions were active in all three monocots studied. Best results were obtained for a construct having both a transcriptional and a translational fusion as well as intron and exon sequences (PORCEHyg). The level of GUS activity was in the range of activities as obtained by the 35S CaMV promoter transcriptionally fused to gusA. The gusA fusion with the light-inducible gene (GOS5) was active in green seedling tissues of all monocots studied. Also a weak expression compared to the GOS2 constructs was found in stably transformed rice callus. The gusA fusions with the mannopine synthase promoters 1' and 2' of the TR-DNA were transiently expressed at lower levels in cell suspensions than PORCEHyg. For stably transformed rice callus the expression of the GOS2-gusA fusion often decreased during prolonged subculture. This decrease in GUS activity and the various GUS-staining phenotypes of transgenic calli are explained by the presence of different cell types in the suspensions used and in the calli. It is presumed that the nature of the cells and their relative contribution in the calli change drastically upon further subculture.
Asunto(s)
Clonación Molecular , Genes de Plantas , Glucuronidasa/genética , Hordeum/genética , Lolium/genética , Oryza/genética , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , ADN , Glucuronidasa/biosíntesis , Datos de Secuencia Molecular , Fenotipo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Biosíntesis de Proteínas , Mapeo Restrictivo , Transcripción Genética , Transformación GenéticaRESUMEN
A novel DNA-binding activity, designated CBF, has been identified in nuclear extracts from tobacco leaf, stem and root tissue. CBF interacts specifically with a 30 bp promoter fragment, referred to as cyt-1, of the Agrobacterium tumefaciens T-DNA cytokinin (T-cyt) gene. The T-cyt promoter, although of bacterial origin is active in planta and the 30 bp cyt-1 element is located within a region that is essential for T-cyt promotor activity in leaf, stem and root cells of tobacco plants. Gel retardation assays using different synthetic oligonucleotides and methylation interference experiments pinpointed the binding site of CBF to a GC-rich sequence ATGCCCCACA within the cyt-1 element. Site-directed mutagenesis of the CBF binding site within the T-cyt promoter by using PCR resulted in an almost complete loss of T-cyt promoter activity in transgenic tobacco plants. In a gain-of-function experiment a hexamer of cyt-1 was shown to be able to confer leaf, stem and root expression when fused upstream of a TATA box containing -55 derivative of the T-cyt promoter. A mutant cyt-1 hexamer, defective in CBF binding, did not show activity above background levels. These results indicate that binding of CBF to the cyt-1 element is required for cyt-1 directed gene expression, suggesting that CBF might act as a transcriptional activator. Apart from the ASF-1 binding site of the CaMV 35S promoter, which is also present in the T-DNA nopaline and octopine synthase genes, the cyt-1 element is the only other identified element reported until now that in combination with a TATA box is sufficient to drive gene expression in multiple tobacco tissue types.
Asunto(s)
Agrobacterium tumefaciens/genética , ADN Bacteriano/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Neoplasias , Nicotiana/genética , Nicotiana/metabolismo , Plantas Tóxicas , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Factores de Unión al Sitio Principal , Expresión Génica , Genes de Plantas , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Nicotiana/microbiologíaRESUMEN
A novel selection system for plant genetic transformation was developed based on the enzyme tryptophan decarboxylase (TDC; EC 4.1.1.28) from Catharanthus roseus. This enzyme converts the toxic tryptophan analogue 4-methyl tryptophan (4-mT) into the non-toxic compound 4-methyl tryptamine. Expression of tdc in transgenic plants that have no endogenous TDC-activity allows selection on 4-mT. A vector was constructed containing a tdc cDNA clone under control of the constitutively expressed cauliflower mosaic virus 35S promoter. This vector was used in Agrobacterium-mediated tobacco leaf disc transformation experiments. The optimal concentration for selection with 4-mT was found to be 0.1 mM. The transformed nature of shoots obtained after tdc gene transfer and subsequent selection on 0.1 mM 4-mT was confirmed by northern blot analysis.
Asunto(s)
Descarboxilasas de Aminoácido-L-Aromático/genética , Marcadores Genéticos , Nicotiana/genética , Plantas Tóxicas , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Clonación Molecular , Kanamicina/farmacología , Plantas Modificadas Genéticamente , Mapeo Restrictivo , Nicotiana/citología , Nicotiana/enzimología , Transformación Genética , Triptófano/análogos & derivados , Triptófano/farmacologíaRESUMEN
The promoter region of the Agrobacterium tumefaciens T-cyt gene was fused to a beta-glucuronidase (gusA) reporter gene and introduced into tobacco plants. Detection of gusA expression in transgenic F1 progeny revealed that the T-cyt promoter is active in many, if not all, cell types in leaves, stems and roots of fully developed plants. Developmental stage-dependent promoter activity was observed in seedlings. Analysis of 5'-deleted promoter fragments showed that sequences located between positions -185 and -139 with respect to the T-cyt translational start codon are essential for T-cyt promoter activity in transfected tobacco protoplasts as well as in transformed tobacco plants.
Asunto(s)
Agrobacterium tumefaciens/genética , Citocininas/genética , Nicotiana/microbiología , Plantas Tóxicas , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Clonación Molecular , ADN Bacteriano , Plantas Modificadas Genéticamente , Protoplastos , Eliminación de Secuencia , Nicotiana/genética , TransfecciónRESUMEN
Transcriptional and translational fusions between the reading frame of the beta-D-glucuronidase gene (gusA) and the 2' as well as the 1' promoter of mannopine synthase (mas), a TR locus of Agrobacterium tumefaciens, were made. The expression of these constructs was studied in the transgenic F1 offspring of independent tobacco transformants at the protein level by assaying for GUS activity and western blot analysis of the GUS protein and at the steady-state mRNA level. In leaves, stems and roots no correlation was found between steady-state levels of GUS mRNA and enzyme activity. In older tissues significantly higher GUS activities were found. This is explained by the stable character of the GUS protein together with an accumulation of protein upon ageing. Three to ten times higher GUS activities were found for in vitro grown plants than for greenhouse-grown plants of the same offspring, despite similar levels of GUS mRNA. Roots from in vitro grown plants display three to ten times higher GUS activities than stems and leaves. In transgenic plants grown in vitro, containing a translational fusion with two AUGs in phase, the initiation of translation in leaf material occurred at both AUGs. Initiation of translation at the first AUG, however, was ten times more frequent. In contrast, initiation in roots from in vitro grown plants occurred exclusively at the second AUG.
Asunto(s)
Agrobacterium tumefaciens/genética , Regulación Enzimológica de la Expresión Génica , Glucuronidasa/biosíntesis , Hidroliasas/genética , Nicotiana/genética , Plantas Tóxicas , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Transcripción Genética , Agrobacterium tumefaciens/enzimología , Secuencia de Bases , Southern Blotting , Western Blotting , ADN/genética , ADN/aislamiento & purificación , Glucuronidasa/genética , Hidroliasas/biosíntesis , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Plantas Modificadas Genéticamente , Sistemas de Lectura , Mapeo Restrictivo , Nicotiana/enzimologíaRESUMEN
A single copy gene has been isolated, termed GOS2, from rice. Sequence comparison revealed highly similar genes in mammals and yeast, indicating that GOS2 encodes an evolutionary conserved protein. GOS2 mRNA was detected in all tissues examined. When the upstream region was translationally fused to the reporter gene gusA it was found to drive expression in a variety of rice tissues and in cell suspensions of other monocot species following introduction by particle bombardment. Therefore, the GOS2 promoter is potentially useful for genetic engineering of monocots. A DNA-binding activity from rice, termed rice ASF-1, with similar binding specificity as the cloned tobacco transcription factor TGA-1a, was found to bind to a TGACG sequence motif in the GOS2 promoter. Possible roles for rice ASF-1 in the transcriptional activation of the GOS2 promoter are discussed.
Asunto(s)
Genes de Plantas/genética , Proteínas Nucleares/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Regiones Promotoras Genéticas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Ingeniería Genética , Glucuronidasa/genética , Datos de Secuencia Molecular , Plantas/genética , Proteínas de Unión al ARN , Proteínas Recombinantes/biosíntesis , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Factores de Empalme Serina-Arginina , Distribución TisularRESUMEN
Agrobacterium tumefaciens transfers part of its Ti plasmid, the transferred DNA (T-DNA), to plant cells during tumor induction. Expression of this T-DNA in plant cells results in their transformation into tumor cells. There are similarities between the process of T-DNA transfer to plants and the process of bacterial conjugation. Here, the T-DNA transfer machinery mediated conjugation between bacteria. Thus, products of the Vir region of the Ti plasmid of Agrobacterium tumefaciens, normally involved in transfer of DNA from bacteria to plants, can direct the conjugative transfer of an IncQ plasmid between agrobacteria.
RESUMEN
The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) converts tryptophan into tryptamine, and thereby channels primary metabolites into indole alkaloid biosynthesis. The production of these secondary metabolites in suspension cells of Catharanthus roseus depends on medium composition. Of the possible variables, we investigated the effect of hormones on the expression of the tdc gene in cell cultures. Omission of NAA from the growth medium resulted in accumulation of tdc mRNA. The addition of 1-naphthaleneacetic acid (NAA), indoleacetic acid (IAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) rapidly reduced the enhanced tdc transcript level. Cytokinin was unable to suppress the enhanced transcript level. Hairy roots transformed by Agrobacterium rhizogenes also showed a reduction of the tdc mRNA level after NAA addition. Run-off transcription experiments showed that the down-regulation takes place at the transcriptional level within 15 minutes and independent of de novo protein synthesis. Thus one of the mechanisms which control the activity of terpenoid indole alkaloid biosynthesis in C. roseus cell cultures is the negative regulation by auxin of the gene involved in the first committed step.
Asunto(s)
Descarboxilasas de Aminoácido-L-Aromático/genética , Regulación hacia Abajo/efectos de los fármacos , Ácidos Indolacéticos/farmacología , Plantas/enzimología , Transcripción Genética/genética , Ácido 2,4-Diclorofenoxiacético/farmacología , Northern Blotting , Células Cultivadas , Citocininas/farmacología , Ácidos Naftalenoacéticos/farmacología , Plantas/efectos de los fármacos , Plantas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Catharanthus roseus (periwinkle) produces a wide range of terpenoid indole alkaloids, including several pharmaceutically important compounds, from the intermediate strictosidine. The complete mRNA sequence for the enzyme strictosidine synthase (SSS) was determined. Comparison of the primary structure of the encoded protein with the amino-terminal sequence of purified SSS indicated the presence of a signal peptide of 31 amino acids in the putative primary translation product. SSS is encoded by a single-copy gene indicating that isoenzymes reported by others are formed post-translationally from a single precursor. The sss gene and the tryptophan decarboxylase gene (tdc), encoding another enzyme essential for indole alkaloid biosynthesis, are coordinately regulated. In plants steady-state mRNA levels are highest in roots. In cell suspension cultures the genes are rapidly down-regulated by auxin. In contrast, both genes are strongly induced by fungal elicitors such as Pythium aphanidermatum culture filtrate or yeast extract. Induction is a rapid, transcriptional event occurring independent of de novo protein synthesis. These results show that a first important regulatory step in the complex process leading to indole alkaloid accumulation in C. roseus suspension cells is transcription of the biosynthetic genes.
Asunto(s)
Alcaloides/metabolismo , Liasas de Carbono-Nitrógeno , Regulación de la Expresión Génica/efectos de los fármacos , Ácidos Indolacéticos/farmacología , Plantas/enzimología , Transferasas/genética , Secuencia de Aminoácidos , Descarboxilasas de Aminoácido-L-Aromático/genética , Secuencia de Bases , Northern Blotting , Southern Blotting , Western Blotting , Quitina/análogos & derivados , Quitina/farmacología , Quitosano , Datos de Secuencia Molecular , Plantas/genética , Conformación Proteica , Señales de Clasificación de Proteína/genética , Salicilatos/farmacología , Ácido SalicílicoRESUMEN
Two rice cDNA clones (COS6 and COS9) were isolated, corresponding to genes that were highly expressed in roots from seedlings and mature plants. A genomic clone (GOS9) corresponding to cDNA clone COS9 was isolated and the intron/exon structure was determined by comparing the nucleotide sequences of the mRNA and the genomic clone. 5' ends and 3' ends of the mRNA were determined by primer extension and S1-nuclease mapping respectively. The open reading frame present in GOS9 potentially encodes a protein (14 kDa) that does not show any significant homology to other proteins in databases.