WRKY is an important complex family of transcription factors involved in plant immune responses. Among them, WRKY70 plays an important role in the process of the plant defense response to the invasion of pathogens. However, the defense mechanism of PsnWRKY70 is not clear in Populus nigra. In this study, we showed that PsnWRKY70-overexpression lines (OE) had fewer leaf blight symptoms than PsnWRKY70-repressing lines (RE). PsnWRKY70 activated MAP kinase cascade genes (PsnM2K4, PsnMPK3, PsnM3K18), calcium channel proteins-related genes (PsnCNG3, PsnCNGC1, PsnCNG4), and calcium-dependent protein kinases genes (PsnCDPKL, PsnCDPKW, PsnCDPKS, PsnCDPKQ). Furthermore, 129 genes of PsnWRKY70 putative genome-wide direct targets (DTGs) were identified by using transcriptome (RNA-seq) and DNA affinity purification sequencing (DAP-seq). PsnWRKY70 directly binds to the promoters of homologous genes and LRR domain proteins to promote the expression of WRKY6, WRKY18, WRKY22, and WRKY22–1, LRR domain proteins LRR8, LRR-RLK, ADR1-like 2, NB-ARC, etc. Our study suggests that PsnWRKY70 enhances the resistance of A. alternata in poplar by activating genes in both pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI).

ATP-binding cassette (ABC) transporters comprise a transport system superfamily which is ubiquitous in eukaryotic and prokaryotic cells. In plants, ABC transporters play important roles in hormone transport and stress tolerance. In this study, 15 BhABC transporters encoded by genes identified from the transcriptome of Betula halophila were categorized into four subfamilies (ABCB, ABCF, ABCG, and ABCI) using structural domain and phylogenetic analyses. Upon B. halophila exposure to exogenous phytohormones and abiotic stressors, gene expression patterns and transcriptional responses for each subfamily of genes were obtained using semi-quantitative RT-PCR analysis. The results demonstrated that expression of most genes belonging to ABCB and ABCG subfamilies changed in response to exogenous phytohormone exposures and abiotic stress. These results suggest that BhABC genes may participate in hormone transport and that their expression may be influenced by ABA-dependent signaling pathways involved in abiotic stress responses to various stressors.

The WUSCHEL−related homeobox (WOX) family is a group of plant−specific transcription factors that play important regulatory roles in embryo formation, stem cell stability, and organogenesis. To date, there are few studies on the molecular mechanisms involved in this family of genes in response to stress. Thus, in this study, eight WOX genes were obtained from an endemic Chinese resilient tree species, Populus × xiaohei T. S. Hwang et Liang. Bioinformatic analysis showed that the WOX genes all contained a conserved structural domain consisting of 60 amino acids, with some differences in physicochemical properties. Phylogenetic analysis revealed that WOX members were divided into three evolutionary clades, with four, one, and three members in the ancient, intermediate, and modern evolutionary clades, respectively. The conserved structural domain species as well as the organization and gene structure of WOX genes within the same subfamily were highly uniform. Chromosomal distribution and genome synteny analyses revealed seven segmental−duplicated gene pairs among the PsnWOX gene family that were mainly under purifying selection conditions. Semi−quantitative interpretation (SQ−PCR) analysis showed that the WOX gene was differentially expressed in different tissues, and it was hypothesized that the functions performed by different members were diverse. The family members were strongly and differentially expressed under CdCl₂, NaCl, NaHCO₃, and PEG treatments, suggesting that WOX genes function in various aspects of abiotic stress defense responses. These results provide a theoretical basis for investigating the morphogenetic effects and abiotic stress responses of this gene family in woody plants.

Poplar is an important afforestation and ornamental tree species in Northeast China. The distribution area of saline-alkali land is approximately 765 hm² in Northeast China. The breeding of saline-alkali-resistant transgenic trees could be an effective method of afforestation in saline-alkali land. WRKY transcription factors play a crucial role in abiotic stress. In this study, we analyzed the genetic stability of the two-year-old PsnWRKY70 transgenic poplars. The results showed that PsnWRKY70 of transgenic poplars had been expressed stably and normally at the mRNA level. The gene interference expression (RE) lines had no significant effect on the growth of PsnWRKY70 under NaHCO₃ stress, and the alkali damage index of RE lines was significantly lower than that of WT and overexpression (OE) lines at day 15 under NaHCO₃ stress. POD activity was significantly higher in RE lines than in WT. The MDA content of the RE line was lower than that of the WT line. Transcriptome analysis showed that RE lines up-regulated genes enriched in cell wall organization or biogenesis pathway-related genes such as EXPA8, EXPA4, EXPA3, EXPA1, EXPB3, EXP10, PME53, PME34, PME36, XTH9, XTH6, XTH23, CESA1, CESA3, CES9; FLA11, FLA16 and FLA7 genes. These genes play an important role in NaHCO₃ stress. Our study showed that the interference expression of the PsnWRKY70 gene can enhance the tolerance of NaHCO₃ in poplar.

The ethylene-insensitive 3-like (EIN3/EIL) gene family, as a transcriptional activator in plants, not only plays an important role in the ethylene-signaling pathway in regulating plant growth and development but also participates in the defense against various biotic and abiotic stresses. However, there are few studies on the functions of EIN3/EIL genes in woody plants. Populus × xiaohei is a kind of tree species with strong drought resistance and salt-alkali tolerance and, thus, is an ideal subject for studying abiotic stress mechanisms in trees. Eight EIN3/EIL genes were cloned from Populus × xiaohei. Bioinformatic analysis showed that the PsnEIN3/EIL gene contained a highly conserved EIN3 domain, N-terminal sites rich in proline and glutamine, and other EIN3/EIL family structural characteristics. The results of a multi-species phylogenetic analysis showed that the family EIN3/EIL proteins were divided into three groups (A, B, and C). EIL3 and EIL4 belonged to groups A and B, while EIL2 and EIN3 generally belonged to group C. Analysis of tissue expression characteristics showed that PsnEIN3/EIL was expressed in different tissues and was involved in the development of stem nodes and leaves. The response analysis of the expression of PsnEIN3/EIL under abscisic acid (ABA) and abiotic stresses (salts, heavy metals, alkaline conditions, and drought) showed changes in expression, suggesting that PsnEIN3/EIL may be involved in the processes of plant hormone responses to salts, heavy metals, alkaline conditions, and drought. This study provides a foundation for further elucidation of the functions of EIN3/EIL genes in forest growth and development and abiotic stress responses.