Somatic embryogenesis (SE) is a process by which an embryo is derived from somatic tissue. Transcription factors (TFs) have been identified that control this process. One such TF that promotes SE is AGAMOUS‐like 15 (AGL15). Prior work has shown that AGL15 can both induce and repress gene expression. One way this type of dual function TF works is via protein interactions, so a yeast 2‐hybrid (Y2H) screen was undertaken. One intriguing protein with which AGL15 interacted in Y2H was LBD40. LBD40 encodes a LATERAL ORGAN BOUNDARIES (LOB)‐domain TF that is unique to plants and is primarily expressed during seed development. Here, we confirm the AGL15‐LBD40 interaction by quantitative assays and
The stilbenoid pathway is responsible for the production of resveratrol in grapevine (
- Award ID(s):
- 1916804
- NSF-PAR ID:
- 10367759
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- The Plant Journal
- Volume:
- 110
- Issue:
- 2
- ISSN:
- 0960-7412
- Page Range / eLocation ID:
- p. 529-547
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract in planta co‐immunoprecipation. We also document a role for LBD40, and the closely related protein LBD41, in supporting SE. To determine downstream genes potentially controlled by LBD40, chromatin immunoprecipitation followed by high throughput sequencing (ChIP‐seq) was used. More than 400 binding regions for LBD40 were consistently found genome‐wide. To determine genes responsive to LBD40/41 accumulation, RNA‐seq analysis of transcriptomes of wild‐type control and loss‐of‐functionlbd40/lbd41 was performed. Combining these datasets provides insight into genes directly and indirectly controlled by these LOB domain TFs. The gene ontology (GO) enrichment analysis of these regulated genes showed an overrepresentation of biological processes that are associated with SE, further indicating the importance of LBD40 in SE. This work provides insight into SE, a poorly understood, but essential process to generate transgenic plants to meet agricultural demands or test gene function. This manuscript reports on experiments to understand the role that LDB40, a TF, plays in support of SE by investigating genes directly and indirectly controlled by LBD40 and examining physical and genetic interactions with other TFs active in SE. We uncover targets of LBD40 and an interacting TF of the MADS family and investigate targets involvement in SE. -
Marshall-Colon, Amy (Ed.)Abstract Gene regulatory networks (GRNs) are defined by a cascade of transcriptional events by which signals, such as hormones or environmental cues, change development. To understand these networks, it is necessary to link specific transcription factors (TFs) to the downstream gene targets whose expression they regulate. Although multiple methods provide information on the targets of a single TF, moving from groups of co-expressed genes to the TF that controls them is more difficult. To facilitate this bottom-up approach, we have developed a web application named TF DEACoN. This application uses a publicly available Arabidopsis thaliana DNA Affinity Purification (DAP-Seq) data set to search for TFs that show enriched binding to groups of co-regulated genes. We used TF DEACoN to examine groups of transcripts regulated by treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), using a transcriptional data set performed with high temporal resolution. We demonstrate the utility of this application when co-regulated genes are divided by timing of response or cell-type-specific information, which provides more information on TF/target relationships than when less defined and larger groups of co-regulated genes are used. This approach predicted TFs that may participate in ethylene-modulated root development including the TF NAM (NO APICAL MERISTEM). We used a genetic approach to show that a mutation in NAM reduces the negative regulation of lateral root development by ACC. The combination of filtering and TF DEACoN used here can be applied to any group of co-regulated genes to predict GRNs that control coordinated transcriptional responses.more » « less
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Polen, Tino (Ed.)ABSTRACT Regulation of gene expression is a vital component of cellular biology. Transcription factor proteins often bind regulatory DNA sequences upstream of transcription start sites to facilitate the activation or repression of RNA polymerase. Research laboratories have devoted many projects to understanding the transcription regulatory networks for transcription factors, as these regulated genes provide critical insight into the biology of the host organism. Various in vivo and in vitro assays have been developed to elucidate transcription regulatory networks. Several assays, including SELEX-seq and ChIP-seq, capture DNA-bound transcription factors to determine the preferred DNA-binding sequences, which can then be mapped to the host organism’s genome to identify candidate regulatory genes. In this protocol, we describe an alternative in vitro , iterative selection approach to ascertaining DNA-binding sequences of a transcription factor of interest using restriction endonuclease, protection, selection, and amplification (REPSA). Contrary to traditional antibody-based capture methods, REPSA selects for transcription factor-bound DNA sequences by challenging binding reactions with a type IIS restriction endonuclease. Cleavage-resistant DNA species are amplified by PCR and then used as inputs for the next round of REPSA. This process is repeated until a protected DNA species is observed by gel electrophoresis, which is an indication of a successful REPSA experiment. Subsequent high-throughput sequencing of REPSA-selected DNAs accompanied by motif discovery and scanning analyses can be used for determining transcription factor consensus binding sequences and potential regulated genes, providing critical first steps in determining organisms’ transcription regulatory networks. IMPORTANCE Transcription regulatory proteins are an essential class of proteins that help maintain cellular homeostasis by adapting the transcriptome based on environmental cues. Dysregulation of transcription factors can lead to diseases such as cancer, and many eukaryotic and prokaryotic transcription factors have become enticing therapeutic targets. Additionally, in many understudied organisms, the transcription regulatory networks for uncharacterized transcription factors remain unknown. As such, the need for experimental techniques to establish transcription regulatory networks is paramount. Here, we describe a step-by-step protocol for REPSA, an inexpensive, iterative selection technique to identify transcription factor-binding sequences without the need for antibody-based capture methods.more » « less
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