RESUMEN
Poly-ADP-ribose polymerase (PARP) post-translationally modifies proteins through the addition of ADP-ribose polymers, yet its role in modulating plant development and stress responses is only poorly understood. The experiments presented here address some of the gaps in our understanding of its role in stress tolerance and thereby provide new insights into tolerance mechanisms and growth. Using a combination of chemical and genetic approaches, this study characterized phenotypes associated with PARP inhibition at the physiological level. Molecular analyses including gene expression analysis, measurement of primary metabolites and redox metabolites were used to understand the underlying processes. The analysis revealed that PARP inhibition represses anthocyanin and ascorbate accumulation under stress conditions. The reduction in defense is correlated with enhanced biomass production. Even in unstressed conditions protective genes and molecules are repressed by PARP inhibition. The reduced anthocyanin production was shown to be based on the repression of transcription of key regulatory and biosynthesis genes. PARP is a key factor for understanding growth and stress responses of plants. PARP inhibition allows plants to reduce protection such as anthocyanin, ascorbate or Non-Photochemical-Quenching whilst maintaining high energy levels likely enabling the observed enhancement of biomass production under stress, opening interesting perspectives for increasing crop productivity.
Asunto(s)
Antocianinas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Estrés Fisiológico , Arabidopsis/crecimiento & desarrollo , Vías Biosintéticas/fisiología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Metaboloma/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Fotosíntesis/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Stress caused by environmental factors evokes dynamic changes in plant phenotypes. In this study, we deciphered simultaneously the reaction of plant growth and chlorophyll fluorescence related parameters using a novel approach which combines existing imaging technologies (GROWSCREEN FLUORO). Three different abiotic stress situations were investigated demonstrating the benefit of this approach to distinguish between effects related to (1) growth, (2) chlorophyll-fluorescence, or (3) both of these aspects of the phenotype. In a drought stress experiment with more than 500 plants, poly(ADP-ribose) polymerase (PARP) deficient lines of Arabidopsis thaliana (L.) Heynh showed increased relative growth rates (RGR) compared with C24 wild-type plants. In chilling stress, growth of PARP and C24 lines decreased rapidly, followed by a decrease in Fv/Fm. Here, PARP-plants showed a more pronounced decrease of Fv/Fm than C24, which can be interpreted as a more efficient strategy for survival in mild chilling stress. Finally, the reaction of Nicotiana tabacum L. to altered spectral composition of the intercepted light was monitored as an example of a moderate stress situation that affects chlorophyll-fluorescence related, but not growth-related parameters. The examples investigated in this study show the capacity for improved plant phenotyping based on an automated and simultaneous evaluation of growth and photosynthesis at high throughput.
RESUMEN
Transgenic plants with reduced poly(ADP-ribose) polymerase (PARP) levels have broad-spectrum stress-resistant phenotypes. Both Arabidopsis thaliana and oilseed rape (Brassica napus) lines overexpressing RNA interference-PARP constructs were more resistant to various abiotic stress treatments in laboratory and greenhouse experiments without negative effects on growth, development, and seed production. This outperforming stress tolerance was initially attributed solely to a maintained energy homeostasis due to reduced NAD(+) consumption. We show that in PARP2-deficient Arabidopsis plants, the observed abiotic stress resistance can also be explained by alterations in abscisic acid levels that facilitate the induction of a wide set of defense-related genes.
Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Estrés Oxidativo , Poli(ADP-Ribosa) Polimerasas/genética , Transducción de Señal , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , ADP-Ribosa Cíclica/metabolismo , Genoma de Planta , Análisis por Micromatrices , Modelos Biológicos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Interferencia de ARN , Transducción de Señal/genética , Transcripción GenéticaRESUMEN
Much progress has been made recently in identifying the molecular components of RNA silencing in plants, and in understanding their roles in the biogenesis of small interfering RNAs and microRNAs, in RNA-directed DNA methylation, and in RNA-mediated antiviral defense. However, many crucial questions remain unanswered. What are the molecular bases of sense and antisense transgene-mediated silencing? Why does silencing only appear to spread through transgenes? Plant viruses encode silencing suppressors to counteract host RNA silencing, and some of these suppressors affect microRNA accumulation and function and hence normal plant development. Is viral pathogenicity determined, partly or entirely, by their silencing suppressor activity?
Asunto(s)
Enfermedades de las Plantas/virología , Virus de Plantas/patogenicidad , Interferencia de ARN , Animales , Modelos Genéticos , Plantas Modificadas Genéticamente/genética , ARN de Planta/genéticaRESUMEN
This unit describes the use of satellite tobacco mosaic virus (STMV) vectors in combination with native TMV particles for inducing transient gene silencing in tobacco plants. Target gene fragment selection and insertion, virus delivery procedures, and phenotype screening of silenced plants are described in detail. All critical parameters for tobacco plant cultivation, virus infection, and RNA silencing efficiency are discussed.
Asunto(s)
Silenciador del Gen , Técnicas Genéticas , Vectores Genéticos/genética , Nicotiana/virología , Virus Satélite del Mosaico del Tabaco/genética , Marcación de Gen , Virus Helper/genética , Virus Helper/aislamiento & purificación , Fenotipo , Enfermedades de las Plantas/virología , Hojas de la Planta/genética , Hojas de la Planta/virología , Plantas Modificadas Genéticamente/genética , ARN Viral/genética , ARN Viral/aislamiento & purificación , Nicotiana/genética , Nicotiana/crecimiento & desarrollo , Virus del Mosaico del Tabaco/genética , Virus del Mosaico del Tabaco/aislamiento & purificaciónRESUMEN
We developed a novel, two-component transient gene silencing system in which the satellite tobacco mosaic virus (STMV) is used as vector for the delivery of inhibitory RNA into tobacco plants and the tobacco mosaic virus strain U2 (TMV-U2) is used as helper virus for supplying replication and movement proteins in trans. The main advantage of the system is that by uncoupling virus replication components from silencing induction components, the intensity of silencing becomes more pronounced. We call this system satellite virus-induced silencing system (SVISS) and will demonstrate here its robustness, speed and effectiveness. We were able to obtain pronounced and severe knockout phenotypes for a range of targeted endogenous genes belonging to various biochemical pathways and expressed in different plant tissues, such as genes involved in leaf and flower pigmentation, genes for cell wall synthesis in leaf, stem and root tissues or a ubiquitous RNA polymerase gene. By tandem insertion of more than one target gene sequence into the vector, we were able to induce simultaneous knockouts of an endogenous gene and a transgene. SVISS is the first transient gene silencing system for Nicotiana tabacum, which is a genetically well-characterized bridging species for the Solanaceae plant family.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Genes de Plantas/genética , Genes de Plantas/fisiología , Nicotiana/genética , Vectores Genéticos/genética , Datos de Secuencia Molecular , Fenotipo , Hojas de la Planta/genética , ARN de Planta/genética , ARN de Planta/metabolismo , Virus Satélite del Mosaico del Tabaco/genéticaRESUMEN
In the 'RNA world' hypothesis it is postulated that RNA was the first genetic molecule. Recent discoveries in gene silencing research on plants, fungi and animals show that RNA indeed plays a key role not only in controlling invading nucleic acids, like viruses and transposable elements, but also in regulating the expression of transgenes and endogenous genes. Double-stranded RNAs were identified to be the triggering structures for the induction of a specific and highly efficient RNA silencing system, in which enzyme complexes, like Dicer and RISC, facilitate as 'molecular machines' the processing of dsRNA into characteristic small RNA species. RNA silencing can be transmitted rapidly from silenced to non-silenced cells by short and long distance signaling. There is evidence that at least one component of the signal is a specific, degradation-resistant RNA.