Exploring plant gene regulation: promoters, terminators, and their role in biodesign
Researchers explain how new sequencing methods can and need to be leveraged to benchmark promoters and promoter–terminator combinations in bioengineering
image: (A) Representative structure of a plant promoter. (B and C) Local distribution of short sequences (LDSS)-positive octamer CREs, normalized frequency distribution profiles of CCAAT, Y patch, and Inr, and the percentage of TATA-box-containing promoters at the indicated positions on the dicot (B) and monocot (C) promoters. The images of dicot and monocot CREs were adapted from [14] (Arabidopsis) and [13] (rice), respectively. The images of CCAAT, Y-patch, and Initiator (Inr) were adapted from [6] (soybean, Arabidopsis, poplar, grape, Brachypodium distachyon, sorghum, rice, and maize). The images of TATA-box were adapted from [5] (Arabidopsis and maize).
Credit: BioDesign Research
Plant bioengineering—in which plant genes are modified to include desirable characteristics—is the key to solutions for problems concerning crop resilience and food security. Elements like gene promoters and terminators play a pivotal role in high-precision bioengineering. The interplay between these elements influences transcription and gene expression levels in plants and understanding it is pivotal to achieve precision in plant bioengineering.
In an article that was published on 7th July 2023, in Volume 5 of the journal BioDesign Research, researchers from the United States explain how different promoter–terminator combinations affect gene expression and how new technology can be leveraged to identify and characterize them, thus improving bioengineering tools.
Plant DNA can be fragmented into functional units known as ‘bioparts’ that consist of promoters, terminators, and cis-regulatory elements that orchestrate gene expression. Promoters, with distinct regions like the core, proximal, and distal regions, interact with transcription machinery to regulate gene expression. Promoters have specific motifs, such as TATA-boxes, which impact their function and specificity, or enhancers and repressors, which modulate gene expression patterns in response to varying conditions. Further, cis-regulatory elements in plant promoters act as specific regulators of gene expression. Terminators, on the other hand, mark the end of RNA transcripts, aiding in their processing, transport, and stability while protecting them from degradation. They also prevent read-through transcription of downstream sequences.
Differences exist between the core promoters present in dicot and monocot plants, highlighting their unique features and performances. The rational engineering of synthetic core promoters by combining specific motifs will significantly boost promoter activity. Cis-regulatory elements in plant promoters, found across proximal and distal promoter regions, offer insights into shared transcriptional regulation mechanisms between dicots and monocots.
Previous studies have characterized numerous native plant promoters, curated this information in databases, and utilized it for constitutive or conditional transgene expression. Combinatorial cis-regulatory elements and their interactions with transcription factors determine the strength and expression patterns of these native promoters. Although computational tools aid in motif discovery, experimental validation remains crucial due to prediction limitations.
"Only a small number of promoters and terminators have been experimentally characterized and validated in plants. Thus, there is high demand to expand the number of functionally characterized promoters and terminators to serve as standard biological parts for plant synthetic biology research and bioengineering", explains Professor Wusheng Liu, the lead author of the review article.
The authors touch upon strategies for evaluating and benchmarking promoters and terminators in plant biodesign, focusing on their temporal and spatial expression patterns, environmental responses, and cross-species variation. Methodologies such as transient and stable expression approaches, reporter gene systems, and dual-reporter systems aid in assessing their performance and attributes. "Transient expression approaches are relatively simple and effective and can be quickly completed in various plant cells and tissues/organs. In contrast, stable expression systems involve complex and lengthy stable plant transformation but provide the most robust information on the function and strength of promoters and terminators,” explains Prof. Liu.
Despite challenges in traditional identification methods, sequencing-based approaches like ATAC-seq and the integration of RNA-seq and ATAC-seq offer promising avenues for discovering regulatory elements and refining bioengineering efforts. Benchmarking different regulatory elements with varied expression patterns aids in constructing genetic circuits and pathways in plant biodesign.
In conclusion, the study encapsulates the multifaceted roles of promoters and terminators in plant genetic engineering, showcasing their significance in manipulating gene expression for various applications and highlighting the evolving strategies for their assessment and utilization in plant biodesign.
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References
Authors
Emily G. Brooks1, Estefania Elorriaga1, Yang Liu2,3, James R. Duduit1, Guoliang Yuan2,3, Chung-Jui Tsai3,4,5,6, Gerald A. Tuskan2,3, Thomas G. Ranney7, Xiaohan Yang2,3, and Wusheng Liu1
Affiliations
1. Department of Horticultural Science, North Carolina State University, Raleigh, NC 27607, USA.
2. Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
3. The Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
4. Warnell School of Forestry and Natural Resource, University of Georgia, Athens, GA 30602, USA.
5. Department of Plant Biology, University of Georgia, Athens, GA 30602, USA.
6. Department of Genetics, University of Georgia, Athens, GA 30602, USA.
7. Mountain Crop Improvement Lab, Department of Horticultural Science, Mountain Horticultural Crops Research and Extension Center, North Carolina State University, Mills River, NC 28759, USA.
About Professor Wusheng Liu
Dr. Wusheng Liu is currently an Assistant Professor in the Department of Horticultural Science at North Carolina State University. He has nearly 20 years of research experience and has published over 38 scientific articles with a focus on plant biotechnology and plant synthetic biology. Professor Liu is interested in novel approaches for non-GMO and genotype-independent delivery of the CRISPR/Cas9 system into crops for gene editing, Molecular mechanisms of agronomic traits, crop trait engineering using genetic engineering and gene editing, and computational tool-assisted de novo motif discovery and synthetic promoter engineering.