Drought stress seriously threatens plant growth. The improvement of plant water use efficiency (WUE) and drought tolerance through stomatal regulation is an effective strategy for coping with water shortages. Epidermal patterning factor (EPF)/EPF-like (EPFL) family proteins regulate stomatal formation and development in plants and thus contribute to plant stress adaptation. Here, our analysis revealed the presence of 14 PeEPF members in the Populus euphratica genome, which exhibited a relatively conserved gene structure with 1–3 introns. Subcellular localisation prediction revealed that 9 PeEPF members were distributed in the chloroplasts of P. euphratica, and 5 were located extracellularly. Phylogenetic analysis indicated that PeEPFs can be divided into three clades, with genes within the same clade revealing a relatively conserved structure. Furthermore, we observed the evolutionary conservation of PeEPFs and AtEPF/EPFLs in certain domains, which suggests their conserved function. The analysis of cis-acting elements suggested the possible involvement of PeEPFs in plant response to multiple hormones. Transcriptomic analysis revealed considerable changes in the expression level of PeEPFs during treatment with polyethylene glycol and abscisic acid. The overexpression of PeEPF2 resulted in low stomatal density in transgenetic lines. These findings provide a basis for gaining insights into the function of PeEPFs in response to abiotic stress.

Populus euphratica Oliv. typically has four kinds of heteromorphic leaves: linear (Li), lanceolate (La), ovate (Ov) and broad ovate (Bo). Heteromorphic leaves help P. euphratica adapt to extreme desert environments and further contribute to protection against land desertification in Northwest China. In the authors’ previous research, growth-regulating factors (GRFs) were speculated to be related to the development of P. euphratica heteromorphic leaves via multi-omics integrated analysis. However, the genomic features and biological role of the P. euphratica GRF gene family in heteromorphic leaves are still unclear. In this study, 19 PeGRF genes were genome-widely identified and characterized in P. euphratica, and their physicochemical properties, gene structure and phylogenetic evolution were analyzed. An analysis of the research showed that PeGRFs were unevenly distributed on 11 chromosomes and that PeGRF proteins contained conserved motif 1 (WRC) and motif 2 (QLQ). Moreover, 19, 15, 19 and 22 GRFs were identified in Populus deltoides Marshall, Populus pruinosa Schrenk, Salix sinopurpurea C. Wang et C. Y. Yang and Salix suchowensis W. C. Cheng, respectively. A collinearity analysis showed that the PeGRF family evolved slowly within Populus species. A phylogenetic tree of the GRF family was also constructed, and GRFs were divided into four subfamilies. A large number of cis-acting elements were related to plant growth and development, plant hormone response and stress response on the promoter of PeGRFs. The expression pattern of PeGRFs showed significant up-regulation in broad leaves (Ov and Bo) compared with narrow leaves (Li and La). In combination with the predicted gene regulatory network, PeGRF9 (PeuTF06G01147.1) may have an important contribution to the leaf shape development of P. euphratica. The heterologous expression of PeGRF9 in wild-type plants (Col-0) of Arabidopsis thaliana (L.) Heynh was also studied, showing a significant increase in the leaf area of overexpressing plants compared with the wild type. Nineteen PeGRF gene members were identified and characterized in P. euphratica, and a comparison of the genomic analysis of Populus GRF members revealed their evolutionary features. The further overexpression of PeGRF9 in A. thaliana revealed its biological role in the heteromorphic leaves of P. euphratica. This study not only provides new insights into the evolution and function of PeGRFs in P. euphratica heteromorphic leaves but also helps in an understanding of the adaptive evolution of P. euphratica in drought desert environments.

DNA-binding with one finger (Dof) transcription factors plays critical roles in regulating plant growth and development, as well as modulating responses to biotic and abiotic stresses. While the biological characteristics of the Dof family have been explored across various species, their functions in Populus euphratica remain largely uncharacterized. In this study, we identified 43 PeDof family genes through a genome-wide approach, revealing a total of 10 conserved motifs across all family members. Predictions of cis-acting elements indicated that Dof genes are involved in light signaling, hormone signaling, and stress responses. Phylogenetic analysis classified the 43 Dof genes of P. euphratica into six distinct groups, with genes within the same group exhibiting relatively conserved structures. Expression pattern analyses demonstrated significant regulation of PeDof genes by drought stress, with their expression also being influenced by environmental conditions during seed germination. Furthermore, we identified the Dof gene PeSCAP1, which plays a conserved role in regulating guard cell maturation, underscoring the importance of stomatal morphology and function in leaf water retention. This study enhances our understanding of the role of Dofs in abiotic stress responses and provides valuable insights into their function in Populus euphratica.

DNA methylation, mediated by DNA methyltransferases (DMTs) and demethylases (DMLs), is an important epigenetic modification that maintains genomic stability and regulates gene expression in plant growth, development, and stress responses. However, a comprehensive characterization of these gene families in Populus sect. Turanga remains lacking. In this study, eight PeDMT and two PeDML genes were identified in Populus euphratica, and six PpDMT and three PpDML genes in Populus pruinosa. Phylogenetic analysis revealed that DMTs and DMLs could be classified into four and three subfamilies, respectively. The analysis of cis-acting elements indicated that the promoter regions of both DMTs and DMLs were enriched with elements responsive to growth and development, light, phytohormones, and stress. Collinearity analysis detected three segmentally duplicated gene pairs (PeDMT5/8, PeDML1/2, and PpDML2/3), suggesting potential functional diversification. Transcriptome profiling showed that several PeDMTs and PeDMLs exhibited leaf shape- and developmental stage-specific expression patterns, with PeDML1 highly expressed during early stages and in broad-ovate leaves. Whole-genome bisulfite sequencing revealed corresponding decreases in DNA methylation levels, suggesting that active demethylation may contribute to heteromorphic leaf formation. Overall, this study provides significant insights for exploring the functions and expression regulation of plant DMTs and DMLs and will contribute to future research unraveling the molecular mechanisms of epigenetic regulation in P. euphratica.