Search

Knotted1-like Homeobox (KNOX) proteins play important roles in regulating plant growth, development, and other biological processes. However, little information is available on the KNOX gene family in apple (Malus domestica Borkh.). In this study, 22 KNOX genes were identified in the apple genome. The gene structure, protein characteristics, and promoter region were characterized. The MdKNOX family members were divided into three classes based on their phylogenetic relationships. Quantitative real-time PCR analysis revealed that the majority of MdKNOX genes exhibited strongly preferential expression in buds and were significantly up-regulated during the flower induction period. The transcript levels of MdKNOX genes were responsive to treatments with flowering- and stress-related hormones. The putative upstream regulation factor MdGRF could directly bind to the promoter of MdKNOX15 and MdKNOX19, and inhibit their transcriptional activities, which were confirmed by yeast one-hybrid and dual-luciferase assays. The results provide an important foundation for future analysis of the regulation and functions of the MdKNOX gene family.

[ES] La longevidad de semillas es el tiempo durante el cual las semillas son capaces de germinar. Esta capacidad de germinación se reduce en el tiempo debido a las condiciones de almacenaje, y factores físicos externos como la temperatura y la humedad la reducen en gran medida. No obstante, son múltiples los mecanismos de protección de las semillas para paliar estos daños. Todos estos mecanismos están controlados transcripcionalmente durante el desarrollo de la semilla, y cada especie vegetal sigue su propio programa, causando una variación en la longevidad de las semillas de las diferentes especies. La planta Arabidopsis thaliana se usa mundialmente como especie modelo y dispone de colecciones de mutantes para hacer estudios de genética directa. En uno de estos estudios, realizado en el laboratorio de Ramón Serrano en el IBMCP (UPV-CSIC), se obtuvo un mutante de activation-tagging cuya longevidad de semilla era mayor. Dicho mutante sobreexpresa el gen que codifica el factor de transcripción COG1. El análisis de sus semillas revela una capa de suberina mucho mayor que la de las semillas de las plantas silvestres, y podría ser la explicación de esta mayor longevidad de las semillas debido a una mayor impermeablización ante el agua y el aire, previniendo así eventos oxidativos deletéreos para las semillas. (Bueso, et al., 2016) En este estudio se pretende conocer mejor el papel del factor de transcripción COG1. Concretamente se va a estudiar la regulación transcripcional del gen COG1. Un primer análisis consistirá en localizar la expresión de dicho gen durante el desarrollo de la semilla mediante plantas con el promotor de COG1 unido a la proteína reportera GFP. La segunda estrategia consistirá es buscar factores de transcripción que están implicados en el desarrollo de la cubierta de la semilla y/o en la longevidad de la semilla, que regulen directamente a la región promotora del gen COG1. Para dicha estrategia se usará la técnica yeast one-hybrid en el sistema heterólogo Saccharomyces cerevisiae., [EN] Seed longevity is defined as time during which seeds are able to germinate. This germination capacity decreases over time due to storage conditions. External physical factors such as high temperature and high humidity greatly reduce it. However, there are multiple mechanisms to protect seeds in order to mitigate these damages. All these mechanisms are controlled transcriptionally during seed development, and each plant species follows its own program, causing a variation in seed longevity between different species. Arabidopsis thaliana is used worldwide as a model species and there are different mutant collections to make reverse genetics studies. In one of these studies, conducted in Ramón Serrano’s laboratory at IBMCP (UPV-CSIC), an activating-tagging mutant was obtained whose seed longevity was greater and it overexpresses the transcription factor COG1. Seeds lypid polyester analysis reveals a thicker suberin layer compared to wild-type seeds. This could be the seed greater longevity explanation due to an increase in the impermeability to water and air, thus preventing oxidative events which are deleterious to seeds (Bueso, et al., 2016). In this study, it is intended to better understand the role of COG1 transcription factor. Specifically, COG1 gene transcriptional regulation will be studied. A first analysis will consist of locating the expression of said gene during seed development by plants with COG1 promoter bound to GFP reporter protein. Our second strategy will be to seek for transcription factors that are involved in seed coat development and / or in seed longevity, which directly regulate COG1 gene promoter region. For this strategy, yeast one-hybrid technique will be used in the heterologous Saccharomyces cerevisiae system.

[ES] El tiempo durante el cual las semillas permanecen viables se denomina longevidad de semillas. Esta longevidad de semillas es una característica de alto interés agronómico tanto para la conservación de recursos genéticos en bancos de germoplasma como para la propagación de cultivares. El estudio de la longevidad de semilla es crucial para conocer cómo afectan los factores genéticos y ambientales al deterioro de las semillas y como combatir estos eventos deletéreos que hacen que la semilla no germine. En el laboratorio del profesor Ramón Serrano se han realizado diversos análisis de asociación del genoma completo (GWAS) a partir de más de doscientos ecotipos de la planta Arabidopsis thaliana sujetos a distintos tratamientos de envejecimiento de semilla acelerado. En varios de estos análisis, una región genómica que destaca y podría estar asociada con la longevidad de semilla es la que comprende al factor de transcripción MYB47. Se analizó la longevidad de semilla en líneas de inserción de t-DNA en el gen MYB47 y éstas mostraron tener una menor longevidad. Esto apunta a un importante papel de dicho factor de transcripción en la longevidad de la semilla. El objetivo de este TFG es intentar esclarecer el papel de MYB47 en la red transcripcional que participa en el desarrollo de la semilla. Para ello se han propuesto dos técnicas: analizar la expresión del factor MYB47 y buscar genes que regulan y que son regulados por MYB47. En la primera estrategia se usarán plantas transgénicas con el promotor de MYB47 unido a la proteína reportera GFP para visualizar donde se expresa este factor de transcripción durante el desarrollo de semilla. Para la segunda estrategia se utilizará la técnica yeast one-hybrid en el sistema heterólogo Saccharomices cerevisae, y se buscarán factores de transcripción implicados en el desarrollo de la cubierta de la semilla y/o en la longevidad de la semilla que interaccionen directamente con las regiones promotoras conservadas del gen MYB47. También se contempla clonar regiones promotoras de posibles dianas directas de MYB47 y realizar también este tipo de ensayo de interacción proteína-DNA., [EN] The time during which seeds remain viable is called seed longevity. Longevity of seeds is a characteristic of high agronomic interest both for the conservation of genetic resources in germplasm banks and for the propagation of cultivars. The study of seed longevity is crucial to know how genetic and environmental factors affect the deterioration of seeds and how to combat these deleterious events that cause the seed not to germinate. In the laboratory of Professor Ramón Serrano, several analysis of the complete genome association (GWAS) have been made from more than two hundred ecotypes of the Arabidopsis thaliana plant subject to different accelerated seed aging treatments. In several of these analysis, a genomic region that stands out and could be associated with seed longevity is that which comprises the transcription factor MYB47. The longevity of seed in lines of insertion of t-DNA in the MYB47 gene was analysed and these showed lower longevity. This points to an important role of MYB47 transcription factor in the longevity of the seed. The objective of this TFG is to try to clarify the role of MYB47 in the transcriptional network that participates in the development of the seed. For this, two techniques have been proposed: check the expression of the MYB47 factor during seed development and look for genes that regulate and that are regulated by MYB47. In the first strategy, transgenic plants with a t-DNA insert with the MYB47 promoter bound to the GFP reporter protein will be used to visualize where this transcription factor is expressed during seed development. For the second strategy, the Yeast One-Hybrid technique will be used in the heterologous Saccharomyces cerevisiae system to find transcription factors involved in the development of the seed coat and in the longevity of the seed that interact directly with the conserved promoter regions of the MYB47 gene. It is also contemplated to clone promoter regions of possible direct targets of MYB47 and perform this type of protein-DNA interaction assay.

The aim of this study was to determine how the mulberry (Morus notabilis) polyphenol oxidase 1 gene (MnPPO1) is regulated during plant stress responses by exploring the interaction between its promoter region and regulatory transcription factors. First, we analyzed the cis-acting elements in the MnPPO1 promoter. Then, we used the MnPPO1 promoter region [(1268 bp, including an MYB3R-binding cis-element (MSA)] as a probe to capture proteins in DNA pull-down assays. These analyses revealed that the MYB3R1 transcription factor in M. notabilis (encoded by MnMYB3R1) binds to the MnPPO1 promoter region. We further explored the interaction between the MnPPO1 promoter and MYB3R1 with the dual luciferase reporter, yeast one-hybrid, and chromatin immunoprecipitation assays. These analyses verified that MnMYB3R1 binds to the MSA in the MnPPO1 promoter region. The overexpression of MnMYB3R1 in tobacco upregulated the expression of the tobacco PPO gene. This observation as well as the quantitative real-time PCR results implied that MnMYB3R1 and PPO are involved in the abscisic acid-responsive stress response pathway.

NAC (NAM, ATAF1/2, and CUC2) transcription factors play important roles in many biological processes, and mainly bind to the NACRS with core sequences &ldquo, CACG&rdquo, or &ldquo, CATGTG&rdquo, to regulate gene expression. However, whether NAC proteins can bind to other motifs without these core sequences remains unknown. In this study, we employed a Transcription Factor-Centered Yeast one Hybrid (TF-Centered Y1H) screen to study the motifs recognized by ANAC074. In addition to the NACRS core cis-element, we identified that ANAC074 could bind to MybSt1, NRS1, and NRS2. Y1H and GUS assays showed that ANAC074 could bind the promoters of ethylene responsive genes and stress responsive genes via the NRS1, NRS2, or MybSt1 element. ChIP study further confirmed that the bindings of ANAC074 to MybSt1, NRS1, and NRS2 actually occurred in Arabidopsis. Furthermore, ten NAC proteins from different NAC subfamilies in Arabidopsis thaliana were selected and confirmed to bind to the MybSt1, NRS1, and NRS2 motifs, indicating that they are recognized commonly by NACs. These findings will help us to further reveal the functions of NAC proteins.

Yeast one-hybrid (Y1H) assay has been proven to be a powerful technique to characterize in vivo the interaction between a given transcription factor (TF), or its DNA-binding domain (DBD), and target DNA sequences. Comprehensive characterization of TF/DBD and DNA interactions should allow designing synthetic promoters that would undoubtedly be valuable for biotechnological approaches. Here, we use the ligation-independent cloning system (LIC) in order to enhance the cloning efficiency of DNA motifs into the pHISi Y1H vector. LIC overcomes important limitations of traditional cloning technologies, since any DNA fragment can be cloned into LIC compatible vectors without using restriction endonucleases, ligation, or in vitro recombination.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited., [Background] Using motif detection programs it is fairly straightforward to identify conserved cis-sequences in promoters of co-regulated genes. In contrast, the identification of the transcription factors (TFs) interacting with these cis-sequences is much more elaborate. To facilitate this, we explore the possibility of using several bioinformatic and experimental approaches for TF identification. This starts with the selection of co-regulated gene sets and leads first to the prediction and then to the experimental validation of TFs interacting with cis-sequences conserved in the promoters of these co-regulated genes., [Results] Using the PathoPlant database, 32 up-regulated gene groups were identified with microarray data for drought-responsive gene expression from Arabidopsis thaliana. Application of the binding site estimation suite of tools (BEST) discovered 179 conserved sequence motifs within the corresponding promoters. Using the STAMP web-server, 49 sequence motifs were classified into 7 motif families for which similarities with known cis-regulatory sequences were identified. All motifs were subjected to a footprintDB analysis to predict interacting DNA binding domains from plant TF families. Predictions were confirmed by using a yeast-one-hybrid approach to select interacting TFs belonging to the predicted TF families. TF-DNA interactions were further experimentally validated in yeast and with a Physcomitrella patens transient expression system, leading to the discovery of several novel TF-DNA interactions., [Conclusions] The present work demonstrates the successful integration of several bioinformatic resources with experimental approaches to predict and validate TFs interacting with conserved sequence motifs in co-regulated genes., The work of Z.K., G.H., A.S., F.S., C.B., and L.B. was supported by the STREG project. W.X.’s work was supported by the China Scholarship Council (CSC). The authors would like to acknowledge the support of Jasmin Huebner and Nina Schmidt in the generation and analysis of transgenic A. thaliana.

A combined cross-platform approach is presented to experimentally identify and characterize interactions between mouse transcription factors and regulatory elements at unprecedented resolution and throughput., We generated a mouse-specific transcription factor (TF) library consisting of 750 full-length sequence-verified open-reading frame clones. We used this resource to develop a cross-platform pipeline to experimentally characterize mammalian regulatory elements of interest for interacting TFs at unprecedented throughput and resolution. Using well-described regulatory elements as well as orphan enhancers, we show that this cross-platform pipeline characterizes known and uncovers novel TF–DNA interactions that are relevant in vivo., The comprehensive mapping of gene promoters and enhancers has significantly improved our understanding of how the mammalian regulatory genome is organized. An important challenge is to elucidate how these regulatory elements contribute to gene expression by identifying their trans-regulatory inputs. Here, we present the generation of a mouse-specific transcription factor (TF) open-reading frame clone library and its implementation in yeast one-hybrid assays to enable large-scale protein–DNA interaction detection with mouse regulatory elements. Once specific interactions are identified, we then use a microfluidics-based method to validate and precisely map them within the respective DNA sequences. Using well-described regulatory elements as well as orphan enhancers, we show that this cross-platform pipeline characterizes known and uncovers many novel TF–DNA interactions. In addition, we provide evidence that several of these novel interactions are relevant in vivo and aid in elucidating the regulatory architecture of enhancers.