RESUMEN
The enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15) catalyzes the first committed step in the shikimate pathway of tryptophan synthesis, an important precursor for the production of terpenoid indole alkaloids (TIAs). A full-length cDNA encoding nuclear coded chloroplast-specific DAHP synthase transcript was isolated from a Catharanthus roseus cDNA library. This had high sequence similarity with other members of plant DAHP synthase family. This transcript accumulated in suspension cultured C. roseus cells on ultraviolet (UV-B) irradiation. Pretreatment of C.roseus cells with variety of agents such as suramin, N-acetyl cysteine, and inhibitors of calcium fluxes and protein kinases and MAP kinase prevented this effect of UV-B irriadiation. These data further show that the essential components of the signaling pathway involved in accumulation DAHP synthase transcript in C. roseus cells include suramin-sensitive cell surface receptor, staurosporine-sensitive protein kinase and MAP kinase.
RESUMEN
Suspension cultures of Catharanthus roseus were used to evaluate ultraviolet-B (UV-B) treatment as an abiotic elicitor of secondary metabolites. A dispersed cell suspension culture from C. roseus leaves in late exponential phase and stationary phase were irradiated with UV-B for 5 min. The stationary phase cultures were more responsive to UV-B irradiation than late exponential phase cultures. Catharanthine and vindoline increased 3-fold and 12-fold, respectively, on treatment with a 5-min UV-B irradiation.
RESUMEN
BACKGROUND: Elicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitor-signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cell suspension culture to low dose of UV-B irradiation was found to increase the amount of catharanthine and transcription of genes encoding tryptophan decarboxylase (Tdc) and strictosidine synthase (Str). In the present study, the signaling pathway mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated. RESULTS: Here, we investigate whether cell surface receptors, medium alkalinization, Ca2+ influx, H2O2, CDPK and MAPK play required roles in UV-B signaling leading to enhanced production of catharanthine in C. roseus cell suspension cultures. C. roseus cells were pretreated with various agonists and inhibitors of known signaling components and their effects on the accumulation of Tdc and Str transcripts as well as amount of catharanthine production were investigated by various molecular biology techniques. It has been found that the catharanthine accumulation and transcription of Tdc and Str were inhibited by 3-4 fold upon pretreatment of various inhibitors like suramin, N-acetyl cysteine, inhibitors of calcium fluxes, staurosporine etc. CONCLUSION: Our results demonstrate that cell surface receptor(s), Ca2+ influx, medium alkalinization, CDPK, H2O2 and MAPK play significant roles in UV-B signaling leading to stimulation of Tdc and Str genes and the accumulation of catharanthine in C. roseus cell suspension cultures. Based on these findings, a model for signal transduction cascade has been proposed.
Asunto(s)
Catharanthus/metabolismo , Catharanthus/efectos de la radiación , Transducción de Señal/efectos de la radiación , Rayos Ultravioleta , Alcaloides de la Vinca/biosíntesis , Antineoplásicos/farmacología , Descarboxilasas de Aminoácido-L-Aromático/genética , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Calcio/metabolismo , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas de Unión al Calcio/metabolismo , Liasas de Carbono-Nitrógeno/genética , Liasas de Carbono-Nitrógeno/metabolismo , Catharanthus/genética , Células Cultivadas , Medios de Cultivo/efectos de la radiación , Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/metabolismo , Proteínas Quinasas/metabolismo , Suramina/farmacología , Transcripción Genética/efectos de la radiaciónRESUMEN
The transactivator protein, Tat, is a potential candidate for developing a vaccine against human immunodeficiency virus (HIV-1). Since Tat is not immunodominant, especially when delivered as a genetic vaccine, we expressed codon-optimized subtype-C Tat as a molecular conjugate of ubiquitin, to elicit antigen-specific cell-mediated immune responses. Immunization of mice with different ubiquitin-Tat constructs elicited a strong cellular, but not a humoral, immune response. The combination of codon-optimization and ubiquitin-mediated processing of Tat induced a Th-1 type cellular immune response that was detectable without in vitro stimulation, suggesting its potential utility for destruction of virus-infected cells via CTL-mediated lysis. Preliminary attempts at characterizing the immunodominant regions identified a novel T-helper epitope within the core domain of Tat.