The Identification and Characterization of the PeGRF Gene Family in Populus euphratica Oliv. Heteromorphic Leaves Provide a Theoretical Basis for the Functional Study of PeGRF9

<i>Populus euphratica</i> Oliv. typically has four kinds of heteromorphic leaves: linear (Li), lanceolate (La), ovate (Ov) and broad ovate (Bo). Heteromorphic leaves help <i>P. euphratica</i> 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 <i>P. euphratica</i> heteromorphic leaves via multi-omics integrated analysis. However, the genomic features and biological role of the <i>P. euphratica GRF</i> gene family in heteromorphic leaves are still unclear. In this study, 19 <i>PeGRF</i> genes were genome-widely identified and characterized in <i>P. euphratica</i>, and their physicochemical properties, gene structure and phylogenetic evolution were analyzed. An analysis of the research showed that <i>PeGRFs</i> 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 <i>GRFs</i> were identified in <i>Populus deltoides</i> Marshall, <i>Populus pruinosa</i> Schrenk, <i>Salix sinopurpurea</i> C. Wang et C. Y. Yang and <i>Salix suchowensis</i> W. C. Cheng, respectively. A collinearity analysis showed that the <i>PeGRF</i> 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 <i>PeGRFs</i>. The expression pattern of <i>PeGRFs</i> 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, <i>PeGRF9</i> (<i>PeuTF06G01147.1</i>) may have an important contribution to the leaf shape development of <i>P. euphratica.</i> The heterologous expression of <i>PeGRF9</i> in wild-type plants (Col-0) of <i>Arabidopsis thaliana</i> (L.) Heynh was also studied, showing a significant increase in the leaf area of overexpressing plants compared with the wild type. Nineteen <i>PeGRF</i> gene members were identified and characterized in <i>P. euphratica</i>, and a comparison of the genomic analysis of Populus GRF members revealed their evolutionary features. The further overexpression of <i>PeGRF9</i> in <i>A. thaliana</i> revealed its biological role in the heteromorphic leaves of <i>P. euphratica.</i> This study not only provides new insights into the evolution and function of PeGRFs in <i>P. euphratica</i> heteromorphic leaves but also helps in an understanding of the adaptive evolution of <i>P. euphratica</i> in drought desert environments.