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.

MYB transcription factors containing an R2R3 DNA-binding domain are involved in the regulation of various physiological processes, such as responses to abiotic stresses, including salt stress. In poplar, various MYB transcription factors regulate stress responses. In this study, we aimed to identify the correlation between the transcription factor MYB148 and salt stress responses in the hybrid poplar (Populus alba × P. glandulosa). We induced a mutation in the MYB-binding domain of PagMYB148 using CRISPR/Cas9-mediated editing of the PagMYB148 gene, which induced a more suppressed expression of PagMYB148 than that in the wild-type. Although salt and drought treatments enhance the expression of PagMYB148, the pagmyb148-transgenic plants exhibited more sensitive phenotypes than the wild-type plants under salt stress. After exposure to salinity stress, the chlorophyll content was lower in pagmyb148-transgenic plants than in wild-type plants, whereas the mutation increased ion leakage from cells. Additionally, the expression of genes involved in the salt stress response was higher in pagmyb148-transgenic plants than in the wild-type. After salt treatment, pagmyb148-transgenic plants exhibited an increased level of H₂O₂ and reduced activity of antioxidant enzymes. In summary, the MYB148 transcription factor is involved in the regulation of salt stress resistance in hybrid poplar trees. This report contributes to providing a basis for further investigating the molecular mechanisms of the poplar PagMYB148 transcription factor under abiotic stress.