The ethylene response factor (ERF) family is one of the largest plant-specific transcription factor families, playing an important role in plant development and response to stresses. The ERF76 gene is a member of the poplar ERF transcription factor gene family. First, we validated that the ERF76 gene expressed in leaf and root tissues is responsive to salinity stress. We then successfully cloned the ERF76 cDNA fragment containing an open reading frame from di-haploid Populus simonii × Populus nigra and proved that ERF76 protein is targeted to the nucleus. Finally, we transferred the gene into the same poplar clone by the Agrobacterium-mediated leaf disc method. Using both RNA-Seq and reverse transcription-quantitative polymerase chain reaction, we validated that expression level of ERF76 is significantly higher in transgenic plants than that in the nontransgenic control. Using RNA-Seq data, we have identified 375 genes that are differentially expressed between the transgenic plants and the control under salt treatment. Among the differentially expressed genes, 16 are transcription factor genes and 45 are stress-related genes, both of which are upregulated significantly in transgenic plants, compared with the control. Under salt stress, the transgenic plants showed significant increases in plant height, root length, fresh weight, and abscisic acid (ABA) and gibberellin (GA) concentration compared with the control, suggesting that overexpression of ERF76 in transgenic poplar upregulated the expression of stress-related genes and increased the ability of ABA and GA biosynthesis, which resulted in stronger tolerance to salt stress.

Identification of gene expression patterns of key genes across multiple abiotic stresses is critical for mechanistic understanding of stress resistance in plant. In the present study, we identified differentially expressed genes (DEGs) in di-haploid Populus simonii × P. nigra under respective stresses of NaCl, KCl, CdCl2 and PEG. On the basis of RNA-Seq, we detected 247 DEGs that are shared by the four stresses in wild type poplar, and mRNA abundance of the DEGs were validated in transgenic poplar overexpressing ERF76 gene by RNA-Seq. Results from gene ontology analysis indicated that these genes are enriched in significant pathways, such as phenylpropanoid biosynthesis, phenylalanine metabolism, starch and sucrose metabolism, and plant hormone signal transduction. ERF gene family plays significant role in plant abiotic stresses responses. We also investigated expression pattern of ERF gene family under the four stresses. The ERFs and DEGs share similar expression pattern across the four stresses. The transgenic poplar is superior to wild type in morphologic, physiological and biochemical traits, which demonstrated the ERF76 gene plays a significant role in stress resistance. These studies will give a rise in understanding the stress response mechanisms in poplar.

Extreme environments, especially drought and high salt conditions, seriously affect plant growth and development. Ethylene-responsive factor (ERF) transcription factors play an important role in salt stress response. In this study, a significantly upregulated ERF gene was identified in 84K (Populus alba × P. glandulosa), which was named PagERF072. PagERF072 was confirmed to be a nuclear-localized protein. The results of yeast two-hybrid (Y2H) assay showed that PagERF072 protein exhibited no self-activating activity, and yeast one-hybrid (Y1H) demonstrated that PagERF072 could specifically bind to GCC-box element. Under salt stress, the transgenic poplar lines overexpressing PagERF072 showed improved salt tolerance. The activities of peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) in transgenic poplars were significantly increased relative to those of wild-type (WT) plants, whereas malondialdehyde (MDA) content showed an opposite trend. In addition, reactive oxygen species (ROS) was significantly reduced, and the expression levels of POD- and SOD-related genes were significantly increased in transgenic poplars under salt stress compared with WT. All results indicate that overexpression of the PagERF072 gene can improve the salt tolerance of transgenic poplars.

Background: NAC (NAM/ATAF/CUC) is one of the largest plant-specific transcription factor (TF) families known to play significant roles in wood formation. Acting as master gene regulators, a few NAC genes can activate secondary wall biosynthesis during wood formation in woody plants. Results: In the present study, firstly, we screened 110 differentially expressed NAC genes in the leaves, stems, and roots of di-haploid Populus simonii×P. nigra by RNA-Seq. Then we identified a nucleus-targeted gene, NAC15 gene, which was one of the highly expressed genes in the stem among 110 NAC family members. Thirdly, we conducted expression pattern analysis of NAC15 gene, and observed NAC15 gene was most highly expressed in the xylem by RT-qPCR. Moreover, we transferred NAC15 gene into tobacco and obtained 12 transgenic lines overexpressing NAC15 gene (TLs). And the relative higher content of hemicellulose, cellulose and lignin was observed in the TLs compared to the control lines containing empty vector (CLs). It also showed darker staining in the culms of the TLs with phloroglucinol staining, compared to the CLs. Furthermore, the relative expression level of a few lignin- and cellulose-related genes was significantly higher in the TLs than that in the CLs. Conclusions: The overall results indicated that NAC15 gene is highly expressed in the xylem of poplar and may be a potential candidate gene playing an important role in wood formation in transgenic tobacco.

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.