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

Abstract

Quantitative traits are rarely controlled by a single gene, thereby making multi-gene transformation an indispensable component of modern synthetic biology approaches. However, the shortage of unique gene regulatory elements (GREs) for the robust simultaneous expression of multiple nuclear transgenes is a major bottleneck that impedes the engineering of complex pathways in plants. In this study, we compared the transcriptional efficacies of a comprehensive list of well-documented promoter and untranslated region (UTR) sequences side by side. The strength of GREs was examined by a dual-luciferase assay in conjunction with transient expression in tobacco. In addition, we created suites of new GREs with higher transcriptional efficacies by combining the best performing promoter-UTR sequences. We also tested the impact of elevated temperature and high irradiance on the effectiveness of these GREs. While constitutive promoters ensure robust expression of transgenes, they lack spatiotemporal regulations exhibited by native promoters. Here, we present a proof-of-principle study on the characterization of synthetic promoters based on cis-regulatory elements of three key photorespiratory genes. This conserved biochemical process normally increases under elevated temperature, low CO2, and high irradiance stress conditions and results in ∼25% loss in fixed CO2. To select stress-responsive cis-regulatory elements involved in photorespiration, we analyzed promoters of two chloroplast transporters (AtPLGG1 and AtBASS6) and a key plastidial enzyme, AtPGLP using PlantPAN3.0 and AthaMap. Our results suggest that these motifs play a critical role for PLGG1, BASS6, and PGLP in mediating response to elevated temperature and high-intensity light stress. These findings will not only enable the advancement of metabolic and genetic engineering of photorespiration but will also be instrumental in related synthetic biology approaches.

Plant Synthetic Promoters

“…The obtained promoters possess different tissue-specific, strength, and inducibility properties, enabling the co-expression of numerous genes at different levels, as is required by the complex biosynthesis traits or regulatory circuits responding to various environmental conditions, i.e., biotic, abiotic, tissue-specific, light stress, or hormonal stimulation [184,185,[189][190][191][192][193][194]. To avoid unwanted recombination or homology-dependent gene silencing, the expression of each gene is regulated by a different promoter [189,195].…”

“…Among the analyzed cis-responsive elements, MYB and b-ZIP reduced transgene expression rate, measured by luciferase activity, by recruiting transcriptional repressors. However, bHLH increased luciferase activity in response to elevated temperatures, and AP2 in response to high irradiance [192]. Studies on the role of UTR regions in plants and viruses suggest that plant-derived UTRs outperform viral UTRs under ambient conditions.…”

“…Studies on the role of UTR regions in plants and viruses suggest that plant-derived UTRs outperform viral UTRs under ambient conditions. However, plant-derived UTRs are more sensitive to environmental stress conditions [192].…”

From qualitative to quantitative: the state of the art and challenges for plant synthetic biology

“…Recent studies have also shown the potential for fine‐tuning transgene expression levels by regulatory elements while spatiotemporal specificity is preserved [37,72,77–79]. In order to achieve quantitative expression at various levels, a rational design by combining multiple regulatory elements from various species, such as the core promoters, enhancers, 5′UTR, 3′UTR, and introns, was used to construct expression cassettes with quantitative intensities in multiple species and tissues [54,57,68,80,81]. The theoretical transcriptional activity (TTA) of cassettes with different promoter/terminator combinations is coincident with the experimental activity values in transiently transformed Nicotiana leaves [56].…”

“…Therefore, in addition to the characterization of these elements, redesign new elements that can meet specific requirements is also an indispensable approach. Recent studies have also shown the potential for fine-tuning transgene expression levels by regulatory elements while spatiotemporal specificity is preserved [37, 72,[77][78][79]. In order to achieve quantitative expression at various levels, a rational design by combining multiple regulatory elements from various species, such as the core promoters, enhancers, 5′UTR, 3′UTR, and introns, was used to construct expression cassettes with quantitative intensities in multiple species and tissues [54,57,68,80,81].…”