Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1–sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.

Efficient protoplast isolation and gene transfection remain significant challenges in gymnosperms, particularly in Pinus species, where stable transformation is highly limited. Conventional pine protoplast preparation methods have resulted in extremely low transfection efficiencies, hindering functional genomic studies. This study presents an optimized method for isolating high-yield, viable protoplasts from Pinus densiflora (Korean red pine), providing a robust system for transient gene expression assays. Splitting one-month-old cotyledons produced the highest mesophyll protoplast yield (5.0 × 10⁶ cells/g FW), which further increased to 1.2 × 10⁷ cells/g FW after optimizing the enzyme mixture (4.5% cellulase, 0.7% pectinase, 3% hemicellulase), maintaining viability above 86%. Developing xylem and whole-stem protoplasts were also successfully isolated by mitigating resin leakage and debris contamination, with a 17% sucrose gradient yielding 7.4 × 10⁴ cells/g FW at 81.9% viability. Overcoming prior inefficiencies, this protocol significantly enhances gene transfection efficiency, achieving 94.1% GFP transformation with 82.9% viability. Furthermore, transient activation assays confirmed strong activation of pine-derived reporters by native effectors, underscoring the assay’s suitability for studying gymnosperm-specific gene regulation. Given the limited stable transformation strategies available for Pinus species, this optimized protoplast transient gene expression system provides a practical and reliable platform for transient gene expression analysis, offering valuable opportunities for studying gene function and regulation in gymnosperms.

In conifers, compression wood (CW) with a high lignin content forms at the base of the stem or branch in response to gravity, which is a good model system for studying lignin-rich wood formation. In this study, we identified and characterized the laccase gene family (PdeLAC) in Korean red pine (Pinus densiflora), which is integral to monolignol polymerization. Phylogenetic analysis of 54 PdeLAC genes with those from gymnosperms (i.e., Pinus taeda and Picea abies) and angiosperms (i.e., Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa) revealed their categorization into five groups, highlighting distinct evolutionary relationships compared to angiosperms. Gene structure and motif analysis showed conserved copper-binding loops and variable substrate-binding loops, suggesting functional diversity. Expression profiling indicated that 23 PdeLAC genes, including three (PdeLAC28, PdeLAC1, and PdeLAC31) homologous to AtLAC17, were upregulated in developing xylem during the growing season, particularly in CW. Transgenic poplars overexpressing PdeLAC28 exhibited increased xylem area, cell wall thickness, and Klason lignin content, underscoring its role in lignin biosynthesis and CW formation. This study provides valuable insights into the molecular regulation of lignin biosynthesis in CW of P. densiflora, setting a foundation for advancing our understanding of wood formation mechanisms in gymnosperms.

The genus Pinus (~115 species) represents a cornerstone of boreal and temperate forests and plays a central role in global forestry, industrial applications, and carbon sequestration. Their distinctive biology—including exceptionally large genomes, guaiacyl-rich lignin, tracheid-based xylem, and pronounced seasonal growth regulation—makes pines both scientifically compelling and technically challenging to study. Recent advances in genomics and transcriptomics, supported by emerging multi-omics and computational frameworks, have significantly advanced our understanding of the molecular architecture of wood formation, including key processes such as NAC–MYB regulatory cascades, lignin biosynthesis pathways, and adaptive processes such as compression wood development. Yet functional studies remain limited by low transformation efficiency, regeneration difficulties, and a scarcity of conifer-optimized genetic tools. This review highlights recent breakthroughs in single-cell and spatial transcriptomics, CRISPR-based genome editing, synthetic promoter design, and machine learning-driven regulatory network prediction and comprehensively examines translational applications in biomass improvement, lignin engineering, stress resilience, and industrial biotechnology. By expanding the research frontiers of Pinus, we aim to connect molecular discovery with applied forestry and climate mitigation strategies.