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.

Improving plant water use efficiency (WUE) and drought tolerance by modulating stomatal activity constitutes a promising strategy for mitigating the impacts of water scarcity. SnRK2, a key component of the abscisic acid (ABA) signaling pathway, plays a critical role in modulating stomatal behavior under abiotic stress. However, the functional role of SnRK2 in regulating stomatal movement to enhance WUE and drought tolerance in Populus euphratica remains to be characterized. In this study, 11 PeSnRK2 genes were identified in the P. euphratica genome, each comprising 9–14 exons and exhibiting an uneven distribution across seven chromosomes. Subcellular localization predictions indicated that these proteins are predominantly localized in the Cytoplasm and Cytoskeleton. Phylogenetic analysis grouped the PeSnRK2 genes into three distinct subfamilies, and conserved gene structures were observed within each clade. Analysis of cis-acting regulatory elements suggested that PeSnRK2 genes were involved in hormonal signaling and stress response pathways. Further transcriptomic data also indicated substantial alterations in PeSnRK2 expression due to polyethylene glycol (PEG) and abscisic acid (ABA) treatment. Finally, qRT-PCR and subcellular localization showed that PeSnRK2.6 is highly induced by ABA and functions in both nucleus and cytoplasm. This first characterization in a desert woody species bridged gaps in SnRK2 evolution and function.

NIN-like protein (NLP) transcription factors are key regulators of plant nitrate signaling and stress responses. Although extensively studied in Arabidopsis thaliana and various crops, it has rarely been reported in woody plants, particularly in drought-tolerant tree species. In this study, 10 PeNLP genes were identified in the drought-tolerant tree Populus euphratica Oliv. through comparative genomics. These genes were unevenly distributed across seven chromosomes, and the gene-family expansion was mainly driven by whole-genome duplication (WGD). Analysis of conserved domains showed that PeNLPs contained 4–10 characteristic motifs, and most members possessed the typical RWP-RK and PB1-related domains. Collinearity analysis identified 18 NLP orthologous gene pairs between P. euphratica and its relatives (Populus pruinosa and Salix sinopurpurea), which exceeded the 15 pairs detected between P. euphratica and A. thaliana, indicating that the NLP family is more conserved within the Salicaceae. Phylogenetic analysis divided PeNLPs into three subfamilies, and their promoter regions harbored diverse cis-acting elements associated with hormone signaling, environmental stress, growth, and light response. Transcriptome and qRT-PCR analyses further demonstrated that PeNLPs were generally downregulated under drought stress. Overall, this study systematically characterized the evolution, structure, and drought responsiveness of the PeNLPs, providing a theoretical basis and genetic resources for improving nitrogen use efficiency and drought resistance in trees.

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.

Auxin plays a crucial role in plant growth and development via concentration gradient regulation, with auxin response factors (ARFs) as key transcription factors in its signalling pathway. However, comprehensive identification and characterisation of ARF genes in Salicaceae remain limited. This study performed a genome-wide analysis of ARF genes in three Salicaceae species (Populus euphratica Oliv., Populus pruinosa, and Salix sinopurpurea), aiming to clarify their physicochemical properties, evolutionary relationships, and functional relevance. A total of 34 ARF genes were identified in each species, all being nucleus-localised hydrophilic unstable proteins clustered into six phylogenetic subgroups. Their promoters contain numerous cis-acting elements responsive to light, phytohormones, and stresses. Transcriptome and qRT-PCR data showed significant up-regulation of PeARF18 in ovate/broad-ovate leaves of P. euphratica compared to linear/lanceolate leaves. This study provides preliminary insights into the characterisation and potential role of the Salicaceae ARF gene family, laying a foundation for further functional exploration of PeARF18 in P. euphratica leaf shape development.

Populus euphratica Oliv. serves as a keystone species in desert riparian ecosystems. Owing to its pronounced tolerance to drought and salinity, as well as its robust reproductive capacity, it has become a pioneer species in desert oases. The xyloglucan endotransglucosylase (XET)/hydrolase (XTH) gene family plays a critical role in the remodeling of plant cell walls; however, its potential biological functions in poplar remain poorly understood. In this study, we identified the XTH gene family in P. euphratica and conducted a preliminary functional analysis. A total of 33 PeXTH genes were identified, which were unevenly distributed across the chromosomes, with the highest density observed on chromosome 6. Conserved domain analysis indicated that most members contain the typical GH16 domain associated with xyloglucan endotransglucosylase activity. Phylogenetic analysis classified them into four distinct subgroups, exhibiting evolutionary conservation with the model dicot plant of Arabidopsis thaliana. Notably, the promoter analysis revealed an abundance of ABA-responsive and stress-related cis-elements, suggesting their potential involvement in response to multiple stresses. Under drought stress, PeXTH7 (PeuTF07G00088.1) exhibited a distinct expression pattern, with transcript levels significantly increasing with persistent treatment. RT-qPCR results confirmed that PeXTH7 is highly expressed in both roots and leaves. Furthermore, subcellular localization assays demonstrated that the PeXTH7 protein localizes to the secretory pathway and the cell wall, implying a role in cell wall dynamic remodeling through the regulation of xyloglucan metabolism. The PeXTH7-overexpressing transgenic lines exhibited a significant increase in root length compared to the wild-type controls. As the first systematic analysis of the XTH gene family in P. euphratica, this study fills an important knowledge gap and provides new insights into the adaptive mechanisms of desert tree species.