In plants, a large root system improves the uptake of water and nutrients and is important for responding to drought stress. The poplar WUSCHEL-related homeobox (WOX) transcription factor promotes adventitious rooting, but its regulation of root growth in response to drought stress remains elusive. In this study, we found that PagWOX11/12a from hybrid poplar 84K (Populus alba×Populus glandulosa) is expressed predominantly in the roots and is strongly induced by drought stress. Compared with nontransgenic 84K plants, transgenic poplar plants overexpressing PagWOX11/12a displayed increased root biomass and enhanced drought tolerance, while opposite phenotypes were observed for PagWOX11/12a dominant repression (DR) plants. PagWOX11/12a functions as a nuclear transcriptional activator with a transactivation domain at the C-terminus. In addition, PagERF35 was found to specifically bind to a dehydration-responsive element (DRE) within the PagWOX11/12a promoter and activate PagWOX11/12a gene expression. These results indicate that PagERF35 may activate PagWOX11/12a expression in response to drought stress by promoting root elongation and biomass, thereby increasing drought tolerance of poplar.

Summary The WUSCHEL‐related homeobox (WOX) transcription factors WOX11 and WOX12 regulate adventitious rooting and responses to stress. The underlying physiological and molecular regulatory mechanisms in salt stress tolerance remain largely unexplored. Here, we characterized the roles of PagWOX11/12a from 84K poplar (Populus alba × P. glandulosa) and the underlying regulatory mechanism in salt stress. PagWOX11/12a was strongly induced by salt stress in roots. Overexpression of PagWOX11/12a in poplar enhanced salt tolerance, as evident by the promotion of growth‐related biomass. In contrast, salt‐treated PagWOX11/12a dominant repression plants displayed reduced biomass growth. Under salt stress conditions, PagWOX11/12a‐overexpressed lines showed higher reactive oxygen species (ROS) scavenging capacity and lower accumulation of hydrogen peroxide (H2O2) than non‐transgenic 84K plants, whereas the suppressors displayed the opposite phenotype. In addition, PagWOX11/12a directly bound to the promoter region of PagCYP736A12 and regulated PagCYP736A12 expression. The activated PagCYP736A12 could enhance ROS scavenging, thus reducing H2O2 levels in roots under salt stress in PagWOX11/12a‐overexpressed poplars. The collective results support the important role of PagWOX11/12a in salt acclimation of poplar trees, indicating that PagWOX11/12a enhances salt tolerance through modulation of ROS scavenging by directly regulating PagCYP736A12 expression in poplar.

A highly efficient Agrobacterium-mediated transformation method is needed for the molecular study of model tree species such as hybrid poplar 84K (Populus alba × P. glandulosa cv. ‘84K’). In this study, we report a callus-based transformation method that exhibits high efficiency and reproducibility. The optimized callus induction medium (CIM1) induced the development of calli from leaves with high efficiency, and multiple shoots were induced from calli growing on the optimized shoot induction medium (SIM1). Factors affecting the transformation frequency of calli were optimized as follows: Agrobacterium concentration sets at an OD600 of 0.6, Agrobacterium infective suspension with an acetosyringone (AS) concentration of 100 µM, infection time of 15 min, cocultivation duration of 2 days and precultivation duration of 6 days. Using this method, transgenic plants are obtained within approximately 2 months with a transformation frequency greater than 50%. Polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR) and β-galactosidase (GUS) histochemical staining analyses confirmed the successful generation of stable transformants. Additionally, the calli from leaves were subcultured and used to obtain new explants; the high transformation efficiency was still maintained in subcultured calli after 6 cycles. This method provides a reference for developing effective transformation protocols for other poplar species.