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.

Cadmium (Cd) is a nonessential microelement and low concentration Cd²⁺ has strong toxicity to plant growth. Plant metallothioneins, a class of low molecular, cystein(Cys)-rich and heavy-metal binding proteins, play an important role in both metal chaperoning and scavenging of reactive oxygen species (ROS) with their large number of cysteine residues and therefore, protect plants from oxidative damage. In this study, a metallothionein gene, TaMT3, isolated from Tamarix androssowii was transformed into tobacco (Nicotiana tobacum) through Agrobacterium-mediated leaf disc method, and correctly expressed under the control of 35S promoter. Under Cd²⁺ stress, the transgenic tobacco showed significant increases of superoxide dismutase (SOD) activity and chlorophyll concentration, but decreases of peroxidase (POD) activity and malondialdehyde (MDA) accumulation when compared to the non-transgenic tobacco. Vigorous growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weight were significantly larger than those of the non-transgenic tobacco under Cd²⁺ stress. These results demonstrated that the expression of the exogenous TaMT3 gene increased the ability of ROS cleaning-up, indicating a stronger tolerance to Cd²⁺ stress.

Drought is one of the main environmental factors limiting plant growth and development. The AP2/ERF transcription factor (TF) ERF194 play key roles in poplar growth and drought-stress tolerance. However, the physiological mechanism remains to be explored. In this study, the ERF194-overexpression (OX), suppressed-expression (RNA interference, RNAi), and non-transgenic (WT) poplar clone 717 were used to study the physiology role of ERF194 transcription factor in poplar growth and drought tolerance. Morphological and physiological methods were used to systematically analyze the growth status, antioxidant enzyme activity, malondialdehyde (MDA), soluble sugars, starch, and non-structural carbohydrate (NSC) contents of poplar. Results showed that, compared with WT, OX plants had decrease in plant height, internode length, and leaf area and increased number of fine roots under drought stress. In addition, OX had higher water potential, activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), contents of chlorophyll, soluble sugar, starch, and NSC, implying that ERF194 positively regulates drought tolerance in poplar. The growth status of RNAi was similar to those of WT, but the relative water content and CAT activity of RNAi were lower than those of WT under drought treatment. Based on the transcriptome data, functional annotation and expression pattern analysis of differentially expressed genes were performed and further confirmed by RT-qPCR analysis. Gene ontology (GO) enrichment and gene expression pattern analysis indicated that overexpression of ERF194 upregulated the expression of oxidoreductases and metabolism-related genes such as POD and SOD. Detection of cis-acting elements in the promoters suggested that ERF194 may bind to these genes through MeJA-responsive elements, ABA-responsive elements, or elements involved in defense and stress responses. The above results show that ERF194 improved tolerance to drought stress in poplar by regulating its growth and physiological factors. This study provides a new idea for the role of ERF194 transcription factor in plant growth and drought-stress response.

Research Highlights: Sequence phylogeny, genome organisation, gene structure, conserved motifs, promoter cis-element and expression profiling of poplar NACs related to salt stress were detected. In addition, expression of two salt-induced NACs was analysed. Background and Objectives: NAC transcription factor (TF) proteins are involved in a wide range of functions during plant development and stress-related endurance processes. To understand the function of Populus NAC TFs in salt stress tolerance, we characterised the structure and expression profile of a total of 289 NAC members. Materials and Methods: Sequence phylogeny, genome organisation, gene structure, motif composition and promoter cis-element were detected using bioinformatics. The expression pattern of Populus NAC TFs under salt stress was also detected using RNA-Seq and RT-qPCR. Results: Synteny analysis showed that 46 and 37 Populus NAC genes were involved in whole-genome duplication and tandem duplication events, respectively. The expression pattern of Populus NAC TFs under salt stress showed the expression of the 289 PtNACs of 84K poplar was induced. Similar expression trends of NACs were found in Populus simonii × P. nigra T. S. Hwang et Liang and Arabidopsis thaliana (L.) Heynh. Conclusions: The correlation analysis showed that the expression of two differentially expressed NAC genes PtNAC024 and PtNAC182 was significantly associated with most of the 63 differentially expressed genes tested. The expression of PtNAC024 and PtNAC182 in different tissues was also analysed in silico and different expression patterns were found. Together, this study provides a solid basis to explore stress-related NAC TF functions in Populus salt tolerance and development.