DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques

Background High throughput applications of the reverse transcriptase quantitative PCR (RT-qPCR) for quantification of gene expression demand straightforward procedures to isolate and analyze a considerable number of DNA-free RNA samples. Published protocols are labour intensive, use toxic organic chemicals and need a DNase digestion once pure RNAs have been isolated. In addition, for some tissues, the amount of starting material may be limiting. The convenience of commercial kits is often prohibitive when handling large number of samples. Findings We have established protocols to isolate DNA-free RNA from Arabidopsis thaliana tissues ready for RT-qPCR applications. Simple non-toxic buffers were used for RNA isolation from Arabidopsis tissues with the exception of seeds and siliques, which required the use of organic extractions. The protocols were designed to minimize the number of steps, labour time and the amount of starting tissue to as little as 10–20 mg without affecting RNA quality. In both protocols genomic DNA (gDNA) can be efficiently removed from RNA samples before the final alcohol precipitation step, saving extra purification steps before cDNA synthesis. The expression kinetics of previously characterized genes confirmed the robustness of the procedures. Conclusion Here, we present two protocols to isolate DNA-free RNA from Arabidopsis tissues ready for RT-qPCR applications that significantly improve existing ones by reducing labour time and the use of organic extractions. Accessibility to these protocols is ensured by its simplicity and the low cost of the materials used

La violencia en la pareja desde tres perspectivas: Víctimas, agresores y profesionales

La investigación de problemas sociales complejos como la violencia contra las mujeres dentro de la pareja obliga a buscar mecanismos de observación, análisis e interpretación que sean capaces de explicar acontecimientos ya sucedidos y de anticipar escenarios futuros. El objetivo de este artículo es analizar desde una perspectiva relacional, ecológica y sistémica de la violencia dentro de la pareja los discursos proporcionados por los hombres, las mujeres y los profesionales que intervienen. Para ello se ha planteado un estudio de carácter cualitativo en el que han participado 41 personas (9 víctimas, 18 agresores y 14 profesionales). Los resultados muestran a) la diversidad en las características de hombres y mujeres involucrados en situaciones violentas y en el tipo de relación que establecen, b) la percepción de los participantes del tratamiento diferenciado que las instituciones parecen otorgar a hombres y mujeres, c) la necesidad de revisar algunos de los procedimientos jurídicos y de intervención y d) la importancia de los estados emocionales y su influencia en los comportamientos de victimas, agresores y profesionales. Finalmente se plantean algunas propuestas dirigidas a mejorar los diseños de investigación y las estrategias de intervención

El gen DOFG4 de Arabidopsis participa en la regulación de la germinación de la semilla mediada por ABA

La semilla es un órgano clave en el ciclo de vida de las plantas y su capacidad para permanecer en estado de quiescencia y germinar sólo cuando las condiciones ambientales son favorables ha representado una ventaja evolutiva y permitido el desarrollo de la agricultura. La germinación comienza con una absorción de agua que permite la expansión celular y reiniciar el metabolismo. En sentido estricto, la germinación termina cuando la elongación del eje embrionario permite a la radícula penetrar la cubierta de la semilla. Durante la fase postgerminativa, las sustancias de reserva almacenadas en la semilla son utilizadas para el crecimiento de la plántula hasta que ésta es capaz de fotosintetizar. Los ácidos giberélico (GA) y abscísico (ABA) son las dos hormonas más importantes en el control de la germinación. Diferentes factores ambientales y genéticos son capaces de afectar al balance ABA/GA y el descenso de los niveles de ABA e incremento de los de GA durante la germinación reflejan su papel negativo y positivo, respectivamente, en este proceso. En cereales, los genes de hidrolasas que movilizan las sustancias de reserva responden al GA mediante un motivo en cis tripartito conservado llamado GARC que es reconocido por factores de transcripción (TFs) de las familias MYB (R2R3 y R1) y DOF. Sin embargo, el GARC no parece estar presente en los promotores inducibles por GA en dicotiledóneas (Ogawa et al., 2003) y las diferencias en la respuesta a ABA entre la capa de aleurona de cereales y el endospermo de las dicotiledóneas (Penfield et al., 2006), apunta a la existencia de mecanismos de regulación diferentes. Estamos abordando el estudio de estos mecanismos mediante dos aproximaciones diferentes para identificar y caracterizar elementos en cis y factores de transcripción implicados en la germinación de la dicotiledónea Arabidopsis thaliana

FUSCA3 from barley unveils a common transcriptional regulation of seed-specific genes between cereals and Arabidopsis.

Accumulation of storage compounds in the embryo and endosperm of developing seeds is a highly regulated process that allows seedling growth upon germination until photosynthetic capacity is acquired. A critical regulatory element in the promoters of seed storage protein (SSP) genes from dicotyledonous species is the RY box, a target of B3-type transcription factors. However, the functionality of this motif in the transcriptional regulation of SSP genes from cereals has not been fully established. We report here the identification and molecular characterization of barley FUSCA3, a B3-type transcription factor as yet uncharacterized in monocotyledonous plants. Our results show that both the barley and Arabidopsis FUS3 genes maintain a conserved functionality for the regulation of SSP genes and anthocyanin biosynthesis in these two distantly related phylogenetic groups. Complementation of the loss-of-function mutant fus3 in Arabidopsis by the barley HvFus3 gene resulted in restored transcription from the At2S3 gene promoter and normal accumulation of anthocyanins in the seed. In barley, HvFUS3 participates in transcriptional activation of the endosperm-specific genes Hor2 and Itr1. HvFUS3, which specifically binds to RY boxes in EMSA experiments, trans-activates Hor2 and Itr1 promoters containing intact RY boxes in transient expression assays in developing endosperms. Mutations in the RY boxes abolished the HvFUS3-mediated trans-activation. HvFus3 transcripts accumulate in the endosperm and in the embryo of developing seeds, peaking at mid maturation phase. Remarkably, HvFUS3 interacts with the Opaque2-like bZIP factor BLZ2 in yeast, and this interaction is essential for full trans-activation of the seed-specific genes in plant

Identification of Novel Components of the Unfolded Protein Response in Arabidopsis

Unfavorable environmental and developmental conditions may cause disturbances in protein folding in the endoplasmic reticulum (ER) that are recognized and counteracted by components of the Unfolded Protein Response (UPR) signaling pathways. The early cellular responses include transcriptional changes to increase the folding and processing capacity of the ER. In this study, we systematically screened a collection of inducible transgenic Arabidopsis plants expressing a library of transcription factors for resistance toward UPR-inducing chemicals. We identified 23 candidate genes that may function as novel regulators of the UPR and of which only three genes (bZIP10, TBF1, and NF-YB3) were previously associated with the UPR. The putative role of identified candidate genes in the UPR signaling is supported by favorable expression patterns in both developmental and stress transcriptional analyses. We demonstrated that WRKY75 is a genuine regulator of the ER-stress cellular responses as its expression was found to be directly responding to ER stress-inducing chemicals. In addition, transgenic Arabidopsis plants expressing WRKY75 showed resistance toward salt stress, connecting abiotic and ER-stress responses

A Developmental Switch of Gene Expression in the Barley Seed Mediated by HvVP1 (Viviparous-1) and HvGAMYB Interactions

The accumulation of storage compounds in the starchy endosperm of developing cereal seeds is highly regulated at the transcriptional level. These compounds, mainly starch and proteins, are hydrolyzed upon germination to allow seedling growth. The transcription factor HvGAMYB is a master activator both in the maturation phase of seed development and upon germination, acting in combination with other transcription factors. However, the precise mechanism controlling the switch from maturation to germination programs remains unclear. We report here the identification and molecular characterization of Hordeum vulgare VIVIPAROUS1 (HvVP1), orthologous to ABA-INSENSITIVE3 from Arabidopsis thaliana. HvVP1 transcripts accumulate in the endosperm and the embryo of developing seeds at early stages and in the embryo and aleurone of germinating seeds up to 24 h of imbibition. In transient expression assays, HvVP1 controls the activation of Hor2 and Amy6.4 promoters exerted by HvGAMYB. HvVP1 interacts with HvGAMYB in Saccharomyces cerevisiae and in the plant nuclei, hindering its interaction with other transcription factors involved in seed gene expression programs, like BPBF. Similarly, this interaction leads to a decrease in the DNA binding of HvGAMYB and the Barley Prolamine-Box binding Factor (BPBF) to their target sequences. Our results indicate that the HvVP1 expression pattern controls the full Hor2 expression activated by GAMYB and BPBF in the developing endosperm and the Amy6.4 activation in postgerminative reserve mobilization mediated by GAMYB. All these data demonstrate the participation of HvVP1 in antagonistic gene expression programs and support its central role as a gene expression switch during seed maturation and germination

Quantum Fluctuations for Gravitational Impulsive Waves

Quantum fluctuations for a massless scalar field in the background metric of spherical impulsive gravitational waves through Minkowski and de Sitter spaces are investigated. It is shown that there exist finite fluctuations for de Sitter space.Comment: Submitted to Int. J. Mod. Phys.

The targeted overexpression of SlCDF4 in the fruit enhances tomato size and yield involving gibberellin signalling

[EN] Tomato is one of the most widely cultivated vegetable crops and a model for studying fruit biology. Although several genes involved in the traits of fruit quality, development and size have been identified, little is known about the regulatory genes controlling its growth. In this study, we characterized the role of the tomato SlCDF4 gene in fruit development, a cycling DOF-type transcription factor highly expressed in fruits. The targeted overexpression of SlCDF4 gene in the fruit induced an increased yield based on a higher amount of both water and dry matter accumulated in the fruits. Accordingly, transcript levels of genes involved in water transport and cell division and expansion during the fruit enlargement phase also increased. Furthermore, the larger amount of biomass partitioned to the fruit relied on the greater sink strength of the fruits induced by the increased activity of sucrose-metabolising enzymes. Additionally, our results suggest a positive role of SlCDF4 in the gibberellin-signalling pathway through the modulation of GA(4) biosynthesis. Finally, the overexpression of SlCDF4 also promoted changes in the profile of carbon and nitrogen compounds related to fruit quality. Overall, our results unveil SlCDF4 as a new key factor controlling tomato size and composition.Renau-Morata, B.; Carrillo, L.; Cebolla Cornejo, J.; Molina Romero, RV.; Martí-Renau, R.; Domínguez-Figueroa, J.; Vicente-Carbajosa, J.... (2020). The targeted overexpression of SlCDF4 in the fruit enhances tomato size and yield involving gibberellin signalling. Scientific Reports. 10(1):1-14. https://doi.org/10.1038/s41598-020-67537-x1141011FAO. Crops production database. FAOSTAT. Latest update: 04/03/2020. Food and Agriculture Organization of the United Nations. Rome https://www.fao.org/faostat (2018).Willcox, J. K., Catignani, G. L. & Lazarus, S. Tomatoes and cardiovascular health. Crit. Rev. Food Sci. 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The Arabidopsis Transcription Factor CDF3 Is Involved in Nitrogen Responses and Improves Nitrogen Use Efficiency in Tomato

Nitrate is an essential macronutrient and a signal molecule that regulates the expression of multiple genes involved in plant growth and development. Here, we describe the participation of Arabidopsis DNA binding with one finger (DOF) transcription factor CDF3 in nitrate responses and shows that CDF3 gene is induced under nitrate starvation. Moreover, knockout cdf3 mutant plants exhibit nitrate-dependent lateral and primary root modifications, whereas CDF3 overexpression plants show increased biomass and enhanced root development under both nitrogen poor and rich conditions. Expression analyses of 35S::CDF3 lines reveled that CDF3 regulates the expression of an important set of nitrate responsive genes including, glutamine synthetase-1, glutamate synthase-2, nitrate reductase-1, and nitrate transporters NRT2.1, NRT2.4, and NRT2.5 as well as carbon assimilation genes like PK1 and PEPC1 in response to N availability. Consistently, metabolite profiling disclosed that the total amount of key N metabolites like glutamate, glutamine, and asparagine were higher in CDF3-overexpressing plants, but lower in cdf3-1 in N limiting conditions. Moreover, overexpression of CDF3 in tomato increased N accumulation and yield efficiency under both optimum and limiting N supply. These results highlight CDF3 as an important regulatory factor for the nitrate response, and its potential for improving N use efficiency in crops