The miR169 family is one of the largest families of known miRNAs, which performs important functions in plant growth, development, and responses to biotic/abiotic stresses. However, its functions in response to abiotic stresses are still unclear in poplar. In present study, a total of 33 precursor MIR169s were identified from poplar and divided into 3 groups by evolutionary analysis and multiple sequence alignment, with the members in same group sharing similar motifs. Collinearity analysis revealed miR169s in other species that are homologous to poplar miRNAs. Cis-acting elements predication showed that miR169s may respond to ABA (Abscisic acid) and salt stress, which was verified by qRT-PCR. In addition, 12 pairs of miR169/target gene modules were identified by degradome sequencing and most of these modules responded to ABA and salt stress. Specifically, a part of miRNAs showed opposite expression trends with their targets at a certain period, demonstrating a repressive effect on the target genes. All the results suggest that miR169s perform important functions in response to abiotic stresses in poplar.

The strictosidine synthase-like (SSL) gene family is a small plant immune-regulated gene family that plays a critical role in plant resistance to biotic/abiotic stresses. To date, very little has been reported on the SSL gene in plants. In this study, a total of thirteen SSLs genes were identified from poplar, and these were classified into four subgroups based on multiple sequence alignment and phylogenetic tree analysis, and members of the same subgroup were found to have similar gene structures and motifs. The results of the collinearity analysis showed that poplar SSLs had more collinear genes in the woody plants Salix purpurea and Eucalyptus grandis. The promoter analysis revealed that the promoter region of PtrSSLs contains a large number of biotic/abiotic stress response elements. Subsequently, we examined the expression patterns of PtrSSLs following drought, salt, and leaf blight stress, using RT-qPCR to validate the response of PtrSSLs to biotic/abiotic stresses. In addition, the prediction of transcription factor (TF) regulatory networks identified several TFs, such as ATMYB46, ATMYB15, AGL20, STOP1, ATWRKY65, and so on, that may be induced in the expression of PtrSSLs in response to adversity stress. In conclusion, this study provides a solid basis for a functional analysis of the SSL gene family in response to biotic/abiotic stresses in poplar.

The lignified tissue in the secondary stem is the main source of wood. In this study, we applied RNA-Seq analysis to the poplar stems in three developmental stages, including primary stem (PS), transitional stem (TS), and secondary stem (SS), to identify a total of 2028 genes that were highly expressed in the SS. Gene annotation indicated that the functions of these genes are mainly involved in cell wall biosynthesis, xylem development, and programmed cell death (PCD) processes. Subsequently, we explored the expression pattern of these genes at various developmental stages in the horizontal direction of the wood by ASPwood. The expression of these genes was modularized and correlated with the percentage of lignified xylem, using weighted gene co-expression network analysis (WGCNA). Among the genes, as many as 690 were identified as directly associated with lignification in the SS. In addition, the gene promoter cis-elements and protein interactions were predicted by PlantRegMap and STRING, respectively. The results were introduced into a co-expression network to confirm their relationship. We eventually found 54 TFs dominating this network, of which ADOF1, ATMYB3, AtbZIP44 (Potri.005G231300), ANAC043, ATWRKY40, ATEBP (Potri.010G006800), ARF5, anac075, RAP2.1, ARF16, AT- HSFB3, Potri.014G050000 (from WRKY family), HAT22, AT-HSFB2B, and AtWRKY20 had extremely high connectivity, which may play an important role in the lignification of wood formation at secondary stages.

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.