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

Abstract

Abiotic stresses can cause significant damage to plants. For sustainable bioenergy crop production, it is critical to generate resistant crops to such stress. Engineering promoters to control the precise expression of stress resistance genes is a very effective way to address the problem. Here we developed stably transformed Populus tremula × Populus alba hybrid poplar (INRA 717-1B4) containing one-of-six synthetic drought stress-inducible promoters (SDs; SD9-1, SD9-2, SD9-3, SD13-1, SD18-1, and SD18-3) identified previously by transient transformation assays. We screened green fluorescent protein (GFP) induction in poplar under osmotic stress conditions. Of six transgenic lines containing synthetic promoter, three lines (SD18-1, 9-2, and 9-3) had significant GFP expression in both salt and osmotic stress treatments. Each synthetic promoter employed heptamerized repeats of specific and short cis-regulatory elements (7 repeats of 7-8 bases). To verify whether the repeats of longer sequences can improve osmotic stress responsiveness, a transgenic poplar containing the synthetic promoter of the heptamerized entire SD9 motif (20 bases, containing all partial SD9 motifs) was generated and measured for GFP induction under osmotic stress. The heptamerized entire SD9 motif did not result in higher GFP expression than the shorter promoters consisting of heptamerized SD9-1, 9-2, and 9-3 (partial SD9) motifs. This result indicates that shorter synthetic promoters (~50 bp) can be used for versatile control of gene expression in transgenic poplar. These synthetic promoters will be useful tools to engineer stress-resilient bioenergy tree crops in the future.

Novel synthetic inducible promoters controlling gene expression during water‐deficit stress with green tissue specificity in transgenic poplar

“…To do so, DNA motifs are derived from conserved DNA sequences in native promoters of co-expressed genes of interest in species of interest: typically, non-model plants. Recently, several tandem repeats with unknown DNA motifs have been successfully introduced in transgenic soybean and poplar, and clearly induced downstream genes relating to nematode infection in soybean (Liu et al, 2014;Sultana et al, 2022) and abiotic stresses in poplar (Yang et al, 2021(Yang et al, , 2022b) from transcriptome analysis including na€ ıve microarray analysis. Although these synthetic promoters were activated in transgenic plants by abiotic or biotic stimuli, the responses were not specific to cell types or tissue.…”

“…Agrobacterium-mediated poplar leaf disk transformation with binary plasmids containing a GUS driven by various synthetic promoters LUC coding sequence, in place of RFP, was driven by the CaMV 2 9 35S promoter, while the uidA GUS gene replaced GFP and was driven by one of the designed synthetic promoters (Yang et al, 2022b). Synthetic DNA motif digestion and ligation was performed as described above.…”

“…We showed previously that the synthetic promoter format using head-to-tail repeats of single CRE motifs is effective to induce marker gene expressions in both transient assays and in stable transgenic plants upon induction by stimuli such as high-salt or water-deficit treatments (Yang et al, 2021(Yang et al, , 2022b specificity together with water-stress induction, we utilized the same process as our previous studies to identify DNA motifs of interest as sequence to be repeated in synthetic promoters for poplar. Two kilobase upstream sequences from the ATG initiation codon of co-expressed genes in leaf palisade (98 genes) or vascular tissues (16 genes) were submitted separately to predict wellconserved DNA motifs (Table S4).…”

“…Synthetic promoters are constructed with a transcription factor (TF) that recognizes a sequence containing either a re-formatted CREs or simple repeats of a conserved CRE sequence, as well as a core promoter sequence including the binding site of RNA polymerase II. This format has been employed repeatedly for applications in herbaceous and woody plant species (Cai et al, 2020;Sears et al, 2023;Sultana et al, 2022;Yang et al, 2021Yang et al, , 2022b. Short synthetic promoters composed of head-to-tail repeats of conserved CREs and fused upstream of a À 46 bp 35S promoter can result in a shorter than 100 bp and functional promoter.…”

Genome-Wide Analysis of the Expansin Gene Family in Populus and Characterization of Expression Changes in Response to Phytohormone (Abscisic Acid) and Abiotic (Low-Temperature) Stresses

“…Abiotic stresses, which encompass heat, cold, waterlogging, drought, salt, metals, and nutritional deficiencies, are pressures that plants experience that are not caused by the natural environment. Abiotic stresses significantly impact the growth, development and productivity of trees [32,33]. P. cathayana plants exhibited increased antioxidant activity and reduced growth and photosystem II efficiency under salt stress [34].…”