Rational design and testing of abiotic stress‐inducible synthetic promoters from poplar cis‐regulatory elements

Abstract

Abiotic stress resistance traits may be especially crucial for sustainable production of bioenergy tree crops. Here, we show the performance of a set of rationally designed osmotic-related and salt stress-inducible synthetic promoters for use in hybrid poplar. De novo motif-detecting algorithms yielded 30 water-deficit (SD) and 34 salt stress (SS) candidate DNA motifs from relevant poplar transcriptomes. We selected three conserved water-deficit stress motifs (SD18, SD13 and SD9) found in 16 co-expressed gene promoters, and we discovered a well-conserved motif for salt response (SS16). We characterized several native poplar stress-inducible promoters to enable comparisons with our synthetic promoters. Fifteen synthetic promoters were designed using various SD and SS subdomains, in which heptameric repeats of five-to-eight subdomain bases were fused to a common core promoter downstream, which, in turn, drove a green fluorescent protein (GFP) gene for reporter assays. These 15 synthetic promoters were screened by transient expression assays in poplar leaf mesophyll protoplasts and agroinfiltrated Nicotiana benthamiana leaves under osmotic stress conditions. Twelve synthetic promoters were induced in transient expression assays with a GFP readout. Of these, five promoters (SD18-1, SD9-2, SS16-1, SS16-2 and SS16-3) endowed higher inducibility under osmotic stress conditions than native promoters. These five synthetic promoters were stably transformed into Arabidopsis thaliana to study inducibility in whole plants. Herein, SD18-1 and SD9-2 were induced by waterdeficit stress, whereas SS16-1, SS16-2 and SS16-3 were induced by salt stress. The synthetic biology design pipeline resulted in five synthetic promoters that outperformed endogenous promoters in transgenic plants.

Performance of abiotic stress-inducible synthetic promoters in genetically engineered hybrid poplar (Populus tremula × Populus alba)

“…. This table summarizes previously published results of transient experiments performed by our lab(Yang et al 2021) as well as present results in stably transformed transgenic poplar using the same binary plasmids. Significant GFP increase is denoted by + mark (+: P < 0.05; ++: P < 0.01; -: not detected; n.a.…”

Coordinated transcriptional regulation of the carotenoid biosynthesis contributes to fruit lycopene content in high-lycopene tomato genotypes

“…These genes should be the targets for genetic engineering, gene editing, and marker-assisted breeding to improve fruit lycopene content in tomato. Their promoters could also be engineered using gene editing [ 68 , 69 ] or synthetic biology [ 70 , 71 ] to fine-tune expression of these genes.…”

Cis Elements: Added Boost to the Directed Evolution of Plant Genes

“…[14][15][16] It will be extremely useful to design a reliable strategy for the construction of a more productive gene expression cassette, as has recently been used to generate a highly productive arrangement of cis-elements for an enhanced gene expression. 36 The strategy has already been manually deployed in several recent articles, 37,38 although such a modular script has not been published so far and its utility would be an added boost for improving the expression profile of a plant gene to attain an improved productivity under the natural/industrial stress environment. Subsequent engineering of gene sequence across its active site will then further boost its stability, turnover number, productivity, and lastly an increased crop-yield.…”