Synthetic promoter designs enabled by a comprehensive analysis of plant core promoters

Abstract

Targeted engineering of plant gene expression holds great promise for ensuring food security and for producing biopharmaceuticals in plants. However, this engineering requires thorough knowledge of cis-regulatory elements in order to precisely control either endogenous or introduced genes. To generate this knowledge, we used a massively parallel reporter assay to measure the activity of nearly complete sets of promoters from Arabidopsis, maize and sorghum. We demonstrate that core promoter elements -notably the TATA-box -as well as promoter GC content and promoterproximal transcription factor binding sites influence promoter strength. By performing the experiments in two assay systems, leaves of the dicot tobacco and protoplasts of the monocot maize, we detect species-specific differences in the contributions of GC content and transcription factors to promoter strength. Using these observations, we built computational models to predict promoter strength in both assay systems, allowing us to design highly active promoters comparable in activity to the viral 35S minimal promoter. Our results establish a promising experimental approach to optimize native promoter elements and generate synthetic ones with desirable features.

Toward synthetic plant development

“…Preliminary experiments building similar regulatory programs in bacteria and mammalian cell lines suggests that some of the basic design rules for regulatory circuits may apply in both prokaryotes and eukaryotes ( Tigges et al, 2009 ; Stanton et al, 2014 ). Plus, new molecular tools for controlling transcription and translation in plants, such as synthetic promoters ( Cai et al, 2020 ; Jores et al, 2021 ), polyadenylation signals ( Felippes et al, 2020 ; Andreou et al, 2021 ), and transcription factors ( Schaumberg et al, 2016 ; Belcher et al, 2020 ), are being developed at break neck speeds. Additional tools that improve our control over gene expression in plants will be needed to create synthetic developmental regulation (Outstanding Questions Box).…”

Design and Analysis of Native Photorespiration Gene Motifs of Promoter Untranslated Region Combinations Under Short Term Abiotic Stress Conditions

“…Moreover, recent evidence indicated that AtUBQ10 promoter was more stable and persistent than CaMV 35S in Arabidopsis, tobacco and rice ( Grefen et al, 2010 ; Behera et al, 2015 ; Peramuna et al, 2018 ; Wang et al, 2021 ). While few studies have demonstrated that plant-derived promoters display similar efficacies in driving gene expression in both monocot and dicot species, not all promoters behave similarly ( Christensen et al, 1992 ; Behera et al, 2015 ; Jores et al, 2021 ). This necessitates comparing the activity of plant promoters derived from both monocots and dicots to be tested within an assay.…”

“…However, baring studies by Beringer et al (2017) and Feike et al (2019) , most studies did not compare plant-derived constitutive promoters from various species within a single assay. Besides, plant-derived constitutive promoters, another excellent option to achieve robust and precise expression is the usage of synthetic promoters ( Peramuna et al, 2018 ; Ali and Kim, 2019 ; Cai et al, 2020 ; Jores et al, 2021 ). Since synthetic promoters are assembled linking cis -regulatory elements of various promoter regions upstream of the core promoter (TATA box), one can combine tissue specificity with heightened expression within the same promoter construct.…”

“…Since synthetic promoters are assembled linking cis -regulatory elements of various promoter regions upstream of the core promoter (TATA box), one can combine tissue specificity with heightened expression within the same promoter construct. Unfortunately, unlike yeast or animal genomics, very few studies have been devoted to identifying core cis -regulatory elements underlying developmental and biochemical pathways in plants to help us generate an array of synthetic promoters for gene stacking in crop plants ( Kaplan et al, 2006 ; Kaur et al, 2017 ; Laxa and Fromm, 2018 ; Eseverri et al, 2020 ; Jores et al, 2021 ; Kakei et al, 2021 ). In addition to promoters, upstream and downstream untranslated regions (UTRs) also have a profound effect on gene expression ( Diamos and Mason, 2018 ; Yamamoto et al, 2018 ; de Felippes et al, 2020 ).…”

“…Despite the knowledge of the core photorespiratory enzymes very little is known about how they are regulated ( Rojas et al, 2012 ; Saji et al, 2017 ). Recent reports have shown the potential of utilizing cis -regulatory elements (CREs) of endogenous genes to fine-tune transgene expression for preserving spatiotemporal regulatory characteristics ( Kaur et al, 2017 ; Cai et al, 2020 ; Jores et al, 2021 ; Kakei et al, 2021 ; Melo et al, 2021 ). Synthetic promoters designed with single or multiple CREs identified from native photorespiratory genes can provide valuable insights into the potential positive and negative regulators of photorespiration.…”