Drought has severe effects on plant growth, forest productivity, and survival throughout the world. Understanding the molecular regulation of drought resistance in forest trees can enable effective strategic engineering of novel drought-resistant genotypes of tree species. In this study, we identified a gene, PtrVCS2, encoding a zinc finger (ZF) protein of the ZF-homeodomain transcription factor in Populus trichocarpa (Black Cottonwood) Torr. & A. Gray. ex Hook. Overexpression of PtrVCS2 (OE-PtrVCS2) in P. trichocarpa resulted in reduced growth, a higher proportion of smaller stem vessels, and strong drought-resistance phenotypes. Stomatal movement experiments revealed that the OE-PtrVCS2 transgenics showed lower stomata apertures than wild-type plants under drought conditions. RNA-seq analysis of the OE-PtrVCS2 transgenics showed that PtrVCS2 regulates the expression of multiple genes involved in regulation of stomatal opening and closing, particularly the PtrSULTR3;1-1 gene, and several genes related to cell wall biosynthesis, such as PtrFLA11-12 and PtrPR3-3. Moreover, we found that the water use efficiency of the OE-PtrVCS2 transgenic plants was consistently higher than that of wild type plants when subjected to chronic drought stress. Taken together, our results suggest that PtrVCS2 plays a positive role in improving drought adaptability and resistance in P. trichocarpa.

Drought stress constitutes a persistent constraint on tree survival, and larch, owing to their distinctive needle morphology, deploys drought-responsive strategies that differ from those of broad-leaved trees. In this study, we identified a putative transcription factor, LoWRKY1, that is highly expressed in larch needles and induced by both polyethylene glycol (PEG)-mediated osmotic stress and abscisic acid (ABA) treatment. Heterologous overexpression of LoWRKY1 in tobacco enhanced leaf relative water content (RWC), reduced ion leakage (IL) and malondialdehyde (MDA) levels, thereby mitigating damage to the membrane system. Furthermore, LoWRKY1 was shown to promote superoxide dismutase (SOD) and catalase (CAT) activities in both tobacco and Larix olgensis embryonic callus cells, effectively scavenging reactive oxygen species (ROS) and improving drought tolerance. Collectively, these findings establish that LoWRKY1 plays a positive role in PEG-induced osmotic stress responses in L. olgensis, providing a promising candidate gene for the genetic improvement in the drought tolerance in coniferous trees. More broadly, this work highlights the value of integrating heterologous expression with conifer embryonic callus systems to dissect stress-regulatory networks in conifers, thereby offering a practical strategy to accelerate functional genomics in tree species that remain recalcitrant to stable transformation.