Meng Li

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.

Global forests face increasingly severe drought events, which suppress the growth of larch. Transcription factors (TFs), acting as regulators, can increase plant stress tolerance by modulating gene expression networks. This study integrated RNA-seq, WGCNA and physiology to systematically analyze the adaptation mechanisms of Larix olgensis under drought stress. Among the 50,110 unigenes identified via RNA-seq, 1,391 unigenes were annotated as TFs. These TFs were clustered into four distinct clusters based on expression profiles. TFs within Cluster 1 exhibited significant differential expression during visible wilting of L. olgensis (72 h and 96 h). Ten TFs were selected as candidate TFs from Cluster 1 based on fold-change rank (p < 0.05). Subsequent WGCNA clustered the 50,110 unigenes into 19 co-expression modules. Notably, the firebrick4 module displayed a strong positive correlation with hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents, whereas the darkolivegreen module showed a strong positive correlation with peroxidase (POD) activity. Three TFs in the firebrick4 module (TRINITY_DN13923_c0_g2, TRINITY_DN3004_c0_g1 and TRINITY_DN1230_c0_g1) and two TFs in the darkolivegreen module (TRINITY_DN1156_c1_g3 and TRINITY_DN1156_c1_g1) were identified by co-expression network analysis. The findings of this study contribute to the knowledge of drought-responsive TFs in L. olgensis, establishing a foundational resource for future functional studies and molecular breeding.

Larix olgensis is a valuable timber species in northern China, typically propagated through somatic embryogenesis (SE). However, long-term subculture can lead to a loss of embryogenic potential. This study aimed to establish a simple and stable protocol for the cryopreservation and regeneration of L. olgensis embryogenic callus (EC) that preserves its SE potential and regenerative capacity. The slow-freezing method was employed for cryopreservation. A cryopreservation protocol for L. olgensis EC was developed by optimizing the preculture duration and conditions, cryoprotectant composition and thawing temperature. The results showed that optimal outcomes were achieved using a 24 h stepwise preculture on medium containing 0.2 and 0.4 mol∙L⁻¹ sucrose, followed by cryoprotectant treatment with 0.4 mol∙L⁻¹ sucrose, 2.5% (v/v) dimethyl sulfoxide (DMSO) and 10% polyethylene glycol 6000 (PEG₆₀₀₀), and thawing at 37 °C. EC cryopreserved using this protocol achieved a 100% recovery rate. Moreover, the cryopreserved recoverable EC successfully underwent SE, progressing through germination and rooting. Cryopreservation duration (storage duration in liquid nitrogen) did not affect cell viability and proliferation rate, confirming the protocol’s suitability for long-term cryopreservation of L. olgensis EC. This study provides a valuable reference for the cryopreservation and regeneration of L. olgensis EC, with potential applications for other coniferous species. It establishes a robust foundation for the large-scale propagation of conifers.

Long-term subculture of embryogenic callus leads to a decline in somatic embryogenesis and germination capacity, which may be associated with increased methylation levels. 5-Azacytidine (5-AzaC), a methylation inhibitor, modulates DNA methylation and is widely involved in regulating plant growth, development, and metabolism. In order to investigate the effect of 5-AzaC on somatic embryogenesis and germination in Larix olgensis, we supplemented the proliferation medium with different concentrations of 5-AzaC. The results showed that the addition of 5-AzaC inhibited the proliferation of embryogenic callus, with the proliferation of embryogenic line N2 completely inhibited at 100 μM, while that of embryogenic line N4 ceased at 20 μM. In contrast, treatment with 10 μM and 20 μM of 5-AzaC significantly increased the somatic embryo yield in both embryogenic lines, with the peak yield observed at 20 μM for embryogenic line N2 and at 10 μM for embryogenic line N4. Furthermore, the addition of 10 μM 5-AzaC effectively reduced the deformity rate during somatic embryo germination in embryogenic line N2 and N4, by 15.91% and 13.53%, respectively. These findings demonstrate that 5-AzaC can partially restore the somatic embryogenesis potential of embryogenic callus in L. olgensis under long-term subculture. Additionally, these results suggest that its effects may be both concentration-dependent and genotype-specific. The results provide a potential approach to mitigating the decline in embryogenic competence, while also demonstrating significant potential for large-scale propagation.

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.