CRISPR/Cas9 genome editing technology, particularly cytosine base editing (CBE) systems, emerges as a powerful tool for precise genomic modification in plants, offering transformative applications across agricultural and forestry research and breeding programs. However, current CBE systems in poplar exhibit low efficiency and imprecise base substitutions, and optimization of base editing systems specifically for poplar remains a significant challenge. To address these limitations, we engineer a high-efficiency poplar CBE system (hyPopCBE) by integrating the MS2-UGI system, fusing Rad51 DNA-binding domain, and modifying the nuclear localization signal. Through stepwise optimization, we develop hyPopCBE-V4, which exhibits a synergistic effect in woody plants. Compared to the original hyPopCBE-V1, hyPopCBE-V4 improves C to T editing efficiency while reducing byproducts and exhibiting a narrower editing window. The proportion of plants with clean C to T edits (without byproducts) increases from 20.93% to 40.48%, and the efficiency of clean homozygous C to T editing rises from 4.65% to 21.43%. Using hyPopCBE-V1 and its variants, we induce Pro197Leu mutation in the herbicide target gene PagALS. Poplar lines with edits in all four PagALS homologues exhibit high resistance to tribenuron and nicosulfuron. This study employs a multi-component synergistic optimization strategy that specifically enhances the efficiency and precision of CBE editing in poplar while improving synchronous editing of alleles. Through editing the herbicide resistance gene PagALS, we obtain the herbicide-resistant poplar germplasm. Our research provides a more precise and efficient CBE tool for genetic modification in poplar that can also be applied to other forestry species, demonstrating its potential for advancing forestry research and breeding programs.

Gene expression is regulated by transcription factors binding to cis-elements in promoters. However, efficient cis-elements for genetic engineering are rarely reported. In this study, we identified an 11 bp cis-element in the PtoCP1 promoter that drives strong constitutive gene expression in Populus tomentosa. A 2,270 bp promoter region upstream of the PtoCP1 gene’s translation start site was cloned and named ProPtoCP1. This promoter controls GUS reporter gene expression in the roots, leaves, and stems of Arabidopsis seedlings. Based on the location and density of cis-elements, the PtoCP1 promoter was divided into four fragments by 5′-end deletions. GUS staining and RT-qPCR revealed a key cis-element at −466 to −441 bp essential for gene expression. Further analysis showed that the MYB-TGACG cis-element is a positive regulator, whereas neither MYB nor TGACG alone drove gene expression. This study enhances our understanding of gene expression regulation by cis-elements and provides a valuable tool for genetic engineering.

Protein turnover is essential for cellular metabolism, organelle biogenesis, stress adaptation, and ultimately the viability of cells and tissues. Papain-like cysteine proteases (PLCPs) are one of the vital components in protein degradation. PLCPs have been reported to act in senescence-associated proteolysis, but their roles in vegetative growth remain unclear. We identified PtCP1, an AALP-like PLCP in Populus tomentosa, localized to the vacuole and acid-triggered activated. CRISPR/Cas9-generated loss-of-function mutant (d7) showed dwarfism and non-stomatal photosynthetic limitations. On the other hand, the gain-of-function line (EM, deleted ERFNIN domain) exhibited accelerated growth and enhanced photosynthetic parameters. We showed d7 had the accumulation of Rubisco, which was the most important protein in photosynthetic carbon fixation. Transcriptomics revealed dysregulated carbon metabolism in d7. This data supported PtCP1-mediated proteolysis regulated photosynthetic carbon assimilation via altered Rubisco turnover, and then it increased the biomass accumulation during vegetative growth in woody plants.