Leaf blight is a fungal disease that mainly affects the growth and development of leaves in plants. To investigate the molecular mechanisms of leaf blight defense in poplar, we performed RNA-Seq and enzyme activity assays on the Populus simonii × Populus nigra leaves inoculated with Alternaria alternate fungus. Through weighted gene co-expression network analysis (WGCNA), we obtained co-expression gene modules significantly associated with SOD and POD activities, containing 183 and 275 genes, respectively. We then constructed a co-expression network of poplar genes related to leaf blight resistance based on weight values. Additionally, we identified hub transcription factors (TFs) and structural genes in the network. The network was dominated by 15 TFs, and four out of them, including ATWRKY75, ANAC062, ATMYB23 and ATEBP, had high connectivity in the network, which might play important functions in leaf blight defense. In addition, GO enrichment analysis revealed a total of 44 structural genes involved in biotic stress, resistance, cell wall and immune-related biological processes in the network. Among them, there were 16 highly linked structural genes in the central part, which may be directly involved in poplar resistance to leaf blight. The study explores key genes associated with leaf blight defense in poplar, which further gains an understanding of the molecular mechanisms of biotic stress response in plants.

SIMILAR TO RCD ONE (SRO) gene family is a small plant-specific gene family responsible for growth, development, and stress responses. In particular, it plays a vital role in responding to abiotic stresses such as salt, drought, and heavy metals. Poplar SROs are rarely reported to date. In this study, a total of nine SRO genes were identified from Populus simonii × Populus nigra, which are more similar to dicotyledon SRO members. According to phylogenetic analysis, the nine PtSROs can be divided into two groups, and the members in the same cluster have a similar structure. There were some cis-regulatory elements related to abiotic stress response and hormone-induced factors identified in the promoter regions of PtSROs members. Subcellular localization and transcriptional activation activity of PtSRO members revealed a consistent expression profile of the genes with similar structural profiles. In addition, both RT-qPCR and RNA-Seq results indicated that PtSRO members responded to PEG-6000, NaCl, and ABA stress in the roots and leaves of Populus simonii × Populus nigra. The PtSRO genes displayed different expression patterns and peaked at different time points in the two tissues, which was more significant in the leaves. Among them, PtSRO1c and PtSRO2c were more prominent in response to abiotic stress. Furthermore, protein interaction prediction showed that the nine PtSROs might interact with a broad range of transcription factors (TFs) involved in stress responses. In conclusion, the study provides a solid basis for functional analysis of the SRO gene family in abiotic stress responses in poplar.

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 NAC transcription factor family is well known to play vital roles in plant development and stress responses. For this research, a salt-inducible NAC gene, PsnNAC090 (Po-tri.016G076100.1), was successfully isolated from Populus simonii × Populus nigra. PsnNAC090 contains the same motifs at the N-terminal end of the highly conserved NAM structural domain. The promoter region of this gene is rich in phytohormone-related and stress response elements. Transient transformation of the gene in the epidermal cells of both tobacco and onion showed that the protein was targeted to the whole cell including the cell membrane, cytoplasm and nucleus. A yeast two-hybrid assay demonstrated that PsnNAC090 has transcriptional activation activity with the activation structural domain located at 167–256aa. A yeast one-hybrid experiment showed that PsnNAC090 protein can bind to ABA-responsive elements (ABREs). The spatial and temporal expression patterns of PsnNAC090 under salt and osmotic stresses indicated that the gene was tissue-specific, with the highest expression level in the roots of Populus simonii × Populus nigra. We successfully obtained a total of six transgenic tobacco lines overexpressing PsnNAC090. The physiological indicators including peroxidase (POD) activity, superoxide dismutase (SOD) activity, chlorophyll content, proline content, malondialdehyde (MDA) content and hydrogen peroxide (H₂O₂) content were measured in three transgenic tobacco lines under NaCl and polyethylene glycol (PEG) 6000 stresses. The findings reveal that PsnNAC090 improves salt and osmotic tolerance by enhancing reactive oxygen species (ROS) scavenging and reducing membrane lipid peroxide content in transgenic tobacco. All the results suggest that the PsnNAC090 gene is a potential candidate gene playing an important role in stress response.

ERF family transcription factors are crucial regulators in plants, playing a central role in abiotic stress responses and serving as important targets for stress-tolerant crop breeding. Populus davidiana × P. bolleana, an elite hybrid poplar cultivar artificially selected in northern China, holds significant research value encompassing ecological restoration, economic industries, genetic resource development, and environmental adaptability. This study identified that PdbERF109 expression was significantly upregulated in P. davidiana × P. bolleana response to salt treatment. Furthermore, transgenic poplar lines overexpressing PdbERF109 (OE) were generated. Salt stress assays demonstrated that PdbERF109 overexpression significantly enhanced salt tolerance in transgenic poplar. Compared to wild-type (WT) plants, PdbERF109-OE lines exhibited a significant enhancement in the activities of antioxidant enzymes, with increases of 2.3-fold, 1.2-fold, and 0.5-fold for superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), respectively, while the levels of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) were markedly reduced by 39.89% and 40.03%, indicating significantly enhanced reactive oxygen species (ROS) scavenging capacity and reduced oxidative damage. Concurrently, PdbERF109 overexpression reduced the natural leaf relative water loss (%). Meanwhile, yeast one-hybrid assays confirmed that the PdbERF109 protein specifically binds to GCC-box and DRE cis-acting elements. This study established PdbERF109 as a positive regulator of salt stress responses, highlighting its potential as a target gene for improving plant tolerance to high salinity, providing a promising candidate gene for the molecular breeding of salt-tolerant crops.