Comparative genomic analysis of the Growth Regulating Factors-Interacting Factors (GIFs) in six Salicaceae species and functional analysis of PeGIF3 reveals their regulatory role in Populus heteromorphic leaves

Comparative genomic analysis of the Growth Regulating Factors-Interacting Factors (GIFs) in six Salicaceae species and functional analysis of PeGIF3 reveals their regulatory role in Populus heteromorphic leaves | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative genomic analysis of the Growth Regulating Factors-Interacting Factors (GIFs) in six Salicaceae species and functional analysis of PeGIF3 reveals their regulatory role in Populus heteromorphic leaves Yuqi Yang, Jianhao Sun, Chen Qiu, Peipei Jiao, Zhihua Wu, Zhijun Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3881684/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Mar, 2024 Read the published version in BMC Genomics → Version 1 posted 11 You are reading this latest preprint version Abstract Background The GIF ( Growth-Regulating Factors-Interacting Factors ) gene family plays a vital role in regulating plant growth and development, particularly in controlling leaf, seed, and root meristem homeostasis. As an important adaptative trait of heteromorphic leaves in response to desert environment, however, the regulatory mechanism of heteromorphic leaves by GIF genes in Populus euphratica remains unknown. Results Our study aimed to identify and characterize the GIF genes in Populus euphratica and other five Salicaceae species to investigate their role in regulating heteromorphic leaf development. We identified and characterized a total of 27 GIF genes across six Salicaceae species ( P. euphratica , Populus pruinose , Populus deltoides , Populus trichocarpa , Salix sinopurpurea , and Salix suchowensis ) at the genome-wide level. Then, the comparative genomic analysis among these species suggested that the expansion of GIFs may be derived the specific Salicaceae whole-genome duplication event after their divergence from Arabidopsis. Elements analysis suggested that GIFs were suffering from diverse regulation by hormones and environment clues. Furthermore, the expression data of PeGIFs in heteromorphic leaves, combined with functional information on GIF genes in Arabidopsis thaliana, indicate the role of PeGIFs in regulating leaf development of P. euphratica , especially PeGIFs contain several auxin-related cis-acting elements such as TGA-box. By heterologous expression the PeGIF3 gene in both wild-type plants (Col-0) and gif1 mutant of A. thaliana , a significant difference in leaf expansion along the medial-lateral axis, as well as an increased number of leaf cells, along with the increased number of leaf cells was observed between the overexpressed plants and the wild type. Conclusion The results indicated that PeGIF3 enhances leaf cell proliferation by modulating transcriptional processes, thereby resulting in the expansion of the central-lateral region of the leaf. Our findings not only provide global insights into the evolutionary features of Salicaceae GIFs, but also reveal the regulatory mechanism of PeGIF3 in heteromorphic leaves in P. euphratica . Salicaceae Populus euphratica GIF genes heteromorphic leaves regulatory function Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Salicaceae species are commonly chosen as exemplary forest trees in various research studies due to their ease of vitro regeneration, rapid vegetative reproduction, and significant ecological and economic importance across the northern hemisphere [ 1 – 4 ]. Especially, Populus euphratica characterized by its extraordinary tolerance to both drought and salinity, serves as a pivotal species in desert oases and stands out as an exceptional relic plant thriving in extremely arid environments [ 5 , 6 ]. P. euphratica exhibits a distinct pattern of leaf heteroblasty, with adult trees displaying a sequential production of linear (Li, leaf index ≥ 4), lanceolate (La, 2 ≤ leaf index < 4), ovate (Ov, 1 ≤ leaf index < 2), and broad ovate (Bo, leaf index < 1) leaves as they ascend from the lower to upper canopies, accompanied by a gradual increase in leaf width and area. Previous research has indicated that broad Ov and Bo leaves in P. euphratica exhibit greater resilience to drought compared to narrow Li and La leaves, as evidenced by their thicker palisade tissue and enhanced photosynthetic activity. In addition, there was a significant and positive association observed between leaf area and the content of proline [ 7 , 8 ]. So, it is believed that the presence of heteromorphic leaves in P. euphratica suggests its capacity to adjust to the dry desert environment. Hence, it is crucial to identify and characterize the genes involving in the heteromorphic leaf development of P. euphratica to reveal the functional divergence and adaptive evolution of heteromorphic leaves. Growth-Regulating Factors-Interacting Factors ( GIFs ) represent a class of transcriptional co-activators that collaborate with Growth-Regulating Factors ( GRFs ). Typically, GIFs can interact with GRFs , forming a plant-specific transcriptional complex [ 9 ]. The GIF gene family was initially discovered in Arabidopsis thaliana in 2004[ 10 ]. GIF genes have been intricately linked with plant growth and development [ 11 , 12 ]. In a recent study, it was revealed that GIFs play a vital role in maintaining the precise expression patterns of key developmental factors [ 13 ]. GIF transcriptional coregulators assume the responsibility of regulating the quiescent center organization and the meristem size in A. thaliana [ 14 ]. GIF2 and GIF3 are two additional proteins instrumental in cell proliferation and the development of lateral organs [ 15 ]. The gif2 and gif3 mutations have been associated with the production of smaller lateral organs in comparison to wild-type plant species in A. thaliana [ 15 , 16 ]. Single gif mutant lines in A. thaliana presented a phenotype akin to the control, but the gif triple mutant gif1/gif2/gif3 exhibited an aberrant pistil [ 17 ]. Beyond their roles in model plants, GIFs exert considerable influence in rice [ 18 , 19 ], maize[ 20 ], tomato[ 21 ], tea[ 22 ]. Although GIFs have been investigated in certain plant species, further exploration of this gene family is warranted in Salicaceae. Recent advancements in genomics of Salicaceae species offer an opportunity to characterize the GIF gene family. In this research, we conducted the genome-wide analysis of the GIF gene family, and characterize their structures, conserved motifs, cis-elements, and expression patterns, as well as the comparative genomics analysis of GIF gene family across six different Salicaceae species. We further revealed the role of PeGIF3 in heteromorphic leaf development in P. euphratica through heterologous expression in A. thaliana . These results obtained from this research will offer crucial and precious data for forthcoming investigations concerning the functional characterization of the GIF gene family in Salicaceae species. Results Identification and characterization of GIF s in six Salicaceae species A total of 4, 4, 6, 4, 4, and 5 GIF genes were identified in P. euphratica , P. pruinose , P. deltoides , P. trichocarpa , S. sinopurpurea , and S. suchowensis , respectively. Then, according to the location on chromosomes, the GIF members within the Salicaceae species were assigned names as follows: PeGIF1 to PeGIF4 ; PpGIF1 to PpGIF4 ; PdGIF1 to PdGIF6 ; PtGIF1 to PtGIF4 ; SPUGIF1 to SPUGIF4 ; SSUGIF1 to SSUGIF5 . Almost all of GIF genes were located on single chromosome in six Salicaceae species (Fig. 1). These results suggest that the number of GIF gene family is relatively conserved without dramatic expansion or loss in Salicaceae. Subsequently, we conducted a comprehensive analysis on the characteristics of GIF genes and their encoded proteins (Table 1). The analysis of protein sequences revealed that the GIF proteins have the potential to encode amino acids ranging from 79 to 223, with a molecular weight (MW) varying between 8930.16 and 22222.92 kDa. Additionally, their isoelectric point was observed to fall within the range of 4.71 to 5.93. Table 1 Characteristics of the putative GIF genes. Figure 1. Chromosomal distribution of GIF genes across six Salicaceae species. (A) P. euphratica ; (B) P. pruinose ; (C) P. deltoides ; (D) P. trichocarpa ; (E) S. sinopurpurea ; (F) S. suchowensis . Analysis of phylogenetic and conserved motifs in multi-species of GIFs The evolutionary relationships and potential functions of 30 GIFs from six Salicaceae and one Brassicaceae species were investigated by constructing a phylogenetic tree. The analysis confirmed the classification of GIF genes into three subfamilies, denoted as Group I, II, and III. Specifically, Group I encompassed 13 genes (2 ATGIFs , 2 PeGIFs , 2 PpGIF , 2 PdGIFs , 2 PtGIFs , 2 SPUGIFs , and SSUGIF3 ), Group II comprised 12 genes ( ATGIF1 , PeGIF3 , PpGIF2 , 2 PtGIFs , 2 PdGIFs , 2 SPUGIFs , and 3 SSUGIFs ), while the Group III contained five genes (2 PdGIFs , PpGIF4 , PeGIF2 , and SSUGIF1 ) uniquely belonged to the Salicaceae (Fig. 2A). The unique GIFs from Group III indicated they occurred after the divergence between Arabidopsis and Salicaceae. Then, MEME software was employed to predict conserved motifs in these GIF genes. Five motifs were identified, with motif 1 representing the conserved SSXT(SNH) domain located in the N-terminal region, common among most GIF genes. Notably, motif 3 and motif 4 were present in most genes, while motif 2 exclusively appeared in Group I and Group II genes. Additionally, motif 5 was primarily found in Group II members, hinting at its uniqueness to this group. Interestingly, motifs 2 and 5 at C-terminus were presented in GIF members of only Salicaceae species, possibly indicating their unique roles. Therefore, besides the expanded gene number of GIFs in Salicaceae, the variations of GIF domain numbers may also contribute to the novelty of GIFs in Salicaceae. Figure 2. Analysis of conserved motifs and phylogenetic relationships among GIF genes across seven species. (A) A neighbor-joining (NJ) phylogenetic tree between P. euphratica and other six species followed by conserved motifs. The three groups were marked with different colors on tree branches. (B) The 30 GIF genes in seven species have 5 conserved motifs. Collinearity analysis of multi-species GIFs To further investigate the evolutionary processes of PeGIFs in Populus , we performed the collinear analysis of GIFs between P. euphratica and other 6 species ( A. thaliana, P. pruinose, P. deltoides , P. trichocarpa , S. sinopurpurea and S. suchowensis ). The number of GIF collinear fragements between P. euphratica and other species were as follows: 8 pairs with P. pruinose and S. suchowensis , 12 pairs with P. deltoides and P. trichocarpa , and 5 pairs with S. sinopurpurea , respectively. This result revealed that the collinearity of GIFs within poplar species was more conservative than that between P. euphratica and A. thaliana (5 collinear fragements). Notably, 50 pairs of PeGIF genes exhibited collinear relationships between P. euphratica and the other five Salicaceae species, indicating that these GIF included collinear fragments likely predated the ancestral divergence (Fig. 3). The retention of GIF included collinear fragments possibly resulted from the whole genome duplication event occurred in Salicaceae [ 23 ]. Figure 3. Analysis of collinearity among GIFs involving P. euphratica and six additional species. (A) P. euphratica and A. thaliana (B) P. euphratica and P. pruinose (C) P. euphratica and P. deltoides (D) P. euphratica and P. trichocarpa (E) P. euphratica and S. sinopurpurea (F) P. euphratica and S. suchowensis . The presence of gray lines in the background denotes collinear blocks within P. euphratica and other plant genomes, while the red lines emphasize collinear GIF pairs. Analysis of cis-regulatory elements of GIF Genes To analyze the possible factors influencing the expression patterns of GIF gene family members, we used the PlantCARE online website to predict cis-acting elements. As results, the promoters of almost all GIF s contained various cis-acting elements associated with plant growth and development, phytohormone responses and stress responses. For instance, elements related to plant development, such as CAT-box, TGA-box, AAGAA-motif, GCN4-motif, as-1, Box4 and G-box, etc (Fig. 4A). Among these elements, all genes have Box4 elements, which are part of the conserved DNA module involved in light response. In addition, G-box were important for early senescence of rice flag leaves [ 24 ]. Notably, auxin-related elements (TGA-box) were present in most members of each species, suggesting a role for GIF genes in regulating Populus growth and development. In addition to these elements, there were other elements related to growth and development in some GIF genes, including the CAT-box related to meristem expression, GCN4-motif related to endosperm expression. Meanwhile, stress-related elements encompass anaerobic and anoxic conditions, defense mechanisms against stressors, drought tolerance, low temperature adaptation, and wound response. These elements are regulated by specific cis-acting motifs including antioxidant response element (ARE), GC-rich motif (GC-motif), TC-rich repeats (TCRRs), MYB binding site (MBS), low temperature-responsive element (LTR), and wound-induced promoter motif (WUN-motif). Interestingly, the presence of MYB elements associated with drought stress response was observed in all GIFs , indicating their crucial role in mediating the response to drought-induced stress. Notably, among the hormone-responsive elements, prominent ones included abscisic acid (ABRE), auxin (TGA-element), gibberellin (TATC-box, P-box, GARE-motif), MeJA (TGACG-motif, CGTCA-motif), and salicylic acid (TCA-element). Also, cis-acting elements related to MeJA were the most prevalent among hormone-responsive elements. This analysis suggests that GIF genes participate in diverse growth and developmental processes possibly mediated by hormone signal transduction or environmental stimulus (Fig. 4B). Figure 4. Cis-element analysis of GIF promoters in six Salicaceae species . (A) The numbers and gradient red colors serve as indicators of the abundance of cis-acting elements present in each gene. (B) Color-coded histograms depict the distribution of cis-acting elements in each gene, categorized into three distinct groups. (C) Pie charts depicting the distribution of distinct cis-acting elements within each category. Expression of PeGIFs in heteromorphic leaves from juvenile to adult To investigate the transcriptional regulation of PeGIFs underlying the developmental and functional differentiation processes of heteromorphic leaves, we assessed the expression level on four types of heteromorphic leaves (Li, La, Ov, and Bo) at three different developmental stages (P1, P2, and P3). PeuTF02G01597 ( PeGIF1 ), PeuTF12G00083 ( PeGIF2 ), and PeuTF14G00928 ( PeGIF4 ) exhibited low expression levels. In contrast, PeuTF13G00452(PeGIF3) , which is homologous to ATGIF1 in A. thaliana and plays a crucial role in leaf development, was specifically upregulated in Ov and Bo leaves at the early stage of leaf development (Fig. 5A). Moreover, the expression level of PeGIF3 significantly decreased during later stages of leaf growth (P2-P3), suggesting its potential involvement in promoting broad-leaf expansion during early morphogenesis. Collectively, our results suggest that PeGIF3 plays a dynamic role in regulating the development of broad leaves (Ov and Bo) in P. euphratica , qRT-PCR analysis was further adopted for confirmation of PeGIFs ’ expression in heteromorphic leaves at the P1 leaf stage (Fig. 5B ~ 5C). All the PeGIFs evaluated by qRT-PCR showed the similar expression pattern from RNA-seq result. Collectively, PeGIF3 also encompasses multiple growth-related elements (AAGAA-motif and as-1), particularly auxin-related elements (TGA-box). These results indicated up-regulation of PeGIF3 would regulate the occurrence of broad heteromorphic leaves in P. euphratica . Figure 5. The PeGIFs gene expression patterns in heteromorphic leaves. (A) Expression patterns of PeGIFs across three stages in four heteromorphic leaves; (B) qRT − PCR data on the expression patterns of PeGIF3 for heteromorphic leaves in P1 stage; (C) qRT − PCR data on the expression patterns of other PeGIFs for heteromorphic leaves in P1 stage. Subcellular localization of PeGIF3 To investigate the subcellular distribution of the PeGIF3 protein in plant cells, the GV3101 Agrobacterium strain carrying the 35S: PeGIF3 -YFP construct was introduced into tobacco, and the subcellular localization of PeGIF3 was visualized using a confocal laser scanning microscope. The fluorescence signal of 35S: PeGIF3 -YFP coincides with the nuclear localization signal of NLS-mCherry (Fig. 6). The subcellular localization of PeGIF3 was observed to be predominantly in the nucleus. Figure 6. Fluorescent images of 35S : PeGIF3 -YFP. Bar = 50 mm The function of PeGIF3 involving leaf cell morphology To elucidate the cellular underpinnings of transgenic plant phenotypes, we conducted an analysis on the fifth leaves derived from wild-type plants, atgif1 mutant plants, transgenic wild-type plants overexpressing the PeGIF3 gene, as well as the complementation of PeGIF3 in atgif1 mutant plants (Table 2). Twenty specimens of each plant were selected for sampling. The atgif1 mutant exhibited a more pronounced leaf size defect and a greater reduction in cell number in leaves in this study. To the contrary, the number of cells was significantly higher in overexpressed transgenic plants compared to wild-type plants, indicating a marked increase in cell abundance. The phenotypes of transgenic atgif1 plants expressing the PeGIF3 gene were comparable to those of the wild type (Fig. 7). Table 2. Transgenic plants of this work. Figure 7. The PeGIF3 gene regulates leaf cell morphology. (A) The cross-sectional anatomical structure of the fifth leaf rosette in each plant. (B) The overall morphological characteristics of various plant species. (C) Relevance analysis of cellular quantities across diverse plant species (****, P ≤ 0.0001;ns, P > 0.05). Discussion Heteromorphic leaves in P. euphratica exhibit functional divergence at both physiological and cytological levels. GIF proteins, recognized as key players in leaf development, positively regulate leaf size. Previous investigations have predominantly centered on GIF genes involvement in plant growth and development. This includes their role in maintaining the homeostasis of leaf, seed, and root meristems in A. thaliana [ 13 , 17 , 25 ]. GIF genes have also been associated with modulating tissue and organ size in rice [ 18 , 26 , 27 ]. In maize, they have been implicated in regulating shoot architecture and meristem determinacy [ 20 ]. However, their presence and characteristics in Salicaceae remain unexplored. Recent releases of high-quality genomes for Salicaceae species, including P. euphratica , P. pruinose, P. deltoides , P. trichocarpa , S. sinopurpurea , and S. suchowensis , have opened new avenues for studying the GIF family in Salicaceae. In this study, we identified a total of 27 members belonging to the GIF family within six Salicaceae species, and delve into their structures and phylogenetic relationships within these species. To confirm the evolutionary relationships between GIFs , the 27 GIF members were classified into 3 subfamilies based on domain analysis and phylogenetic tree. The presence of highly conserved domains in the GIF protein within the same group suggests a potential similarity in function. Previously, the ATGIF1 transcription coactivator gene was previously characterized as a positive regulator of cell proliferation in lateral organs, such as leaves and flowers, of A. thaliana [ 15 ]. We hypothesized that members located in the same subfamily as ATGIF1 also function to regulate plant growth. The presence of the highly-conserved SSXT motif was detected in all members comprising the GIF gene family. The findings are in line with prior research [ 10 ]. The N-terminal region of GIF proteins shares similarity with the SNH domain discovered in SYNOVIAL TRANSLOCATION (SYT) in humans, which interacts with BRAHMA (BRM) and BRAHMA RELATED GENE1 (BRG1), two ATPases involved in SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin remodeling processes in human cells. Based on the similarity in sequence, it is probable that GIF transcriptional coactivators facilitate transcription through their interaction with SWI/SNF chromatin remodelers [ 28 ]. Additional motifs were also found in some GIF members, implying that these members might have undergone functional divergence or acquired novel functions throughout the course of plant evolution. Our promoter analysis unveiled a significant number of phytohormone-responsive elements (ABRE, CGTCA, and TGACG), light-responsive elements (G-box, GT1, and Box 4), and stress-responsive elements (ARE, MYB, and MYC) in the promoter regions of these GIF genes. Notably, these GIF gene members have been linked to plant development, a finding consistent with prior research in A. thaliana [ 29 ]. Analysis of gene expression patterns in heteromorphic leaves led to the identification of differentially regulated genes specific to P. euphratica . The expression level of PeGIF3 was found to be significantly higher and exhibited a notable disparity between heteromorphic leaves at the P1 age, which is consistent with the qRT-PCR results. Therefore, we hypothesize that PeGIF3 may be closely regulated in increasing the size of broad leaves early during leaf morphogenesis in P. euphratica . By examining transgenic plants, we observed a significant difference in the number of leaf cells between the overexpressed plants and the wild type. The subsequent observation revealed that PeGIF3 was predominantly observed in the nucleus. It consistent with the conserved motifs analysis of GIFs in multi-species in this study. Therefore, we hypothesized that PeGIF3 enhances leaf cell proliferation by modulating transcriptional processes, thereby resulting in the expansion of the central-lateral region of the leaf. Conclusion In this study, we firstly identified 27 GIF genes in six Salicaceae species and characterize their structures, phylogenetic relationships, conserved motifs and collinearity across Salicaceae species with Arabidopsis as an outgroup. Detailed cis-elements analysis showed the Salicaceae GIFs involved in multiple developmental processes and were regulated by diverse factors, such as phytohormones signals and environmental stimulus. Importantly, only PeGIF3 showed the gradual upregulation along with the development of heteromorphic leaves of Li, La, Ov and Bo, successively. The essential involvement of PeGIF3 in P. euphratica leaf development has been elucidated using RNA-Seq data and qRT-PCR. Further overexpression of PeGIF3 in atgif1 mutant and wild-type of Arabidopsis results in enhanced leaf expansion along the medial-lateral and an increased cell population. Our findings provide a strong foundation for further functional investigations into GIF genes in Salicaceae species and also promote the study of leaf morphological variation among Salicaceae species. Methods Identification characterization of GRF-Interacting Factor homologs in Salicaceae . The PeGIFs were identified based on our P. euphratica genome data [ 30 ]. The hidden Markov model (HMM) profiles for the GIF domain SSXT (PF05030) were acquired from the Pfam protein family database, accessible at http://pfam.xfam.org . HMMER 3.0 ( http://hmmer.org/ ) was employed to conduct a search for potential GIF genes in the six Salicaceae species. The superfluous candidate genes were excluded, and the remaining genes underwent additional validation using SMART ( http://smart.emblheidelberg.de/ ). The protein physicochemical properties of GIF proteins, such as the amino acid count, molecular weight (MW), and theoretical isoelectric point (pI), were determined using the ProtParam tool available at http://web.expasy.org/protparam/ . In addition, the GIFs from five other Salicaceae species were identified based on the genome data of Populus pruinose (National Center for Biotechnology Information(NCBI) with the BioProject accession number PRJNA863418), Populus deltoides (WV94_445) [ 31 ], Populus trichocarpa (V3.1) [ 4 ], Salix sinopurpurea [ 32 ] and Salix suchowensis [ 33 ]. The chromosomal location of GIFs was obtained from the genome annotation files, and the chromosome physical location of the GIF genes was displayed by MapChart V2.32 software. Phylogenetic relationship consensus sequence analysis of multi-species GIFs To investigate the evolutionary relationship among GIF genes, a phylogenetic tree was constructed using the amino acid sequences that encode GIF genes from P. euphratica and various other species. The SMART website was utilized to extract the domain coordinates from the GIF protein sequence of P. euphratica and various other species. The sequences of GIF domain were extracted using the coordinates of the GIF domain and merged into a new sequence matrix. Then, the merged protein sequences were aligned by ClustalW. After aligning the amino acid sequences, gap trimming was performed using the Multiple Alignment Trimming tools of TBtools software with a Site Coverage Cut off parameter set at 0.95. Subsequently, a phylogenetic tree was constructed using MEGA v7 software employing the neighbor-joining (NJ) method with 1000 bootstrap replicates. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches. The Dayhoff matrix-based method was used to calculate evolutionary distances, which were expressed as the number of amino acid substitutions per site. Ambiguous positions were excluded for each pair of sequences using the pairwise deletion option. TBtools and iTOL online website ( https://itol.embl.de/ ) were used to visualize the phylogenetic tree. Additionally, we used the MEME tool ( http://meme-suite.org/ ) to classify and analyze the conserved motifs of each GIF protein sequence. We set the maximum motif number was 5 and other parameters are default settings.. Collinearity analysis of multi-species GIFs The BLASTP alignment was used to identify orthologous pairs between P. euphratica and six other species ( P. pruinose, P. trichocarpa, P. deltoides, S. sinopurpurea, S. suchowensis , and A. thaliana ). Then, the collinear blocks between P. euphratica and each other species of P. deltoide , P. trichocarpa , A. thaliana , S. sinopurpurea and S. sinopurpurea were identified using MCscan software and visualized using JCVI ( https://zenodo.org/record/31631/ ). Promoter analysis of GIF promoters The upstream 2000 base pair (bp) sequences apart from the transcription start sites of these PeGIFs genes were identified as potential promoters using TBtools. Subsequently, the cis -elements within each promoter were identified using PlantCARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) RNA-seq for heteromorphic leaves A total of 12 samples for four leaf shapes in cultivated forests, including linear (Li), lanceolate (La), ovate (Ov) and broad-ovate (Bo) leaves, were collected across the development of leaf age. These samples were collected at various stages of leaf development. Leaf age was categorized into three periods based on field sampling and observation. The first period (P1) was defined as the first day when the leaf blades started unfolding. This was followed by a transitional period (P2) occurring on the 15th day when there was an increase in leaf area. Finally, the third period (P3) occurred on the 30th day when leaves reached maturity. Each type of heteromorphic leaves with different leaf ages was replicated three times for sampling. The napkin was used to delicately clean the leaves, which were then rapidly frozen in liquid nitrogen and stored at an ultra-low temperature of -80℃ in a refrigerator for RNA-seq analysis (the dataset has been made available to the public for access[ 34 ] and preservation through the National Genomics Data Center (NGDC, https://ngdc.cncb.ac.cn/ ), under project number PRJCA005959). Analysis of transcriptomes using short reads from Illumina sequencing. As part of the study, we conducted whole transcriptome sequencing using mRNA-Seq on an Illumina Hiseq X-Ten platform, following the protocol recommended by the vendor. To assess the relative abundance of the annotated genes from P. euphratica , we employed HISAT2 (version 2.0.4) [ 35 ] to align the clean reads against our reference genome. The gene expression was quantified with FPKM using StringTie [ 36 ]. Validation of PeGIFs using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) The heteromorphic foliage was collected from different canopies and stored in an ultra-low temperature refrigerator at -80°C after being rapidly frozen with liquid nitrogen. The procedure followed the methodology described in a previous publication [ 37 ]. Actin gene was used as the endogenous control. Each reaction was performed in biological triplicates, and CT values obtained through qRT-PCR were analyzed using the 2 −ΔΔCT method to calculate relative fold change values. Plant growth conditions, treatments, and sampling All Arabidopsis thaliana mutants and transgenic plants that were used in this study were from the Columbia (Col-0) ecotype. The Arabidopsis seeds were sown on moist soil, stratified at 4 ℃ for 3 days, and then transferred to a growth room with a temperature of 21 ℃ and a photoperiod of 16 hours light/8 hours darkness. Atgif1 (SALK_208834C) seeds were obtained from the AraShare. The leaves of P. euphratica were collected from the forest located at the eastern entrance of Tarim University. Cloning, construction of transgenic plants The laboratory have preserved Escherichia coli (DH5α)、 Agrobacterium tumefaciens (GV3101)、overexpressed vector (pGreenII 0179). The RNA was extracted from P. euphratica Bo leaves using Trizol (Invitrogen), followed by cDNA synthesis using the M5 Sprint qPCR RT kit with gDNA remover (Mei5 Biotechnology). The full-length coding regions of PeGIF3 genes lacking a stop codon were amplified from cDNA, or plasmid using Phanta Max Super-Fidelity DNA polymerase (Vazyme) to ensure high fidelity. Subsequently introduced into a yellow fluorescent protein (YFP) vector to generate a construct using the T4 DNA Ligase (Sangon Biotech). The GIF ::YFP fusion was inserted into the pGreenII 0179 vector, which contained a CaMV 35S promoter and a NOS terminator cassette. The floral dip method was utilized for the transformation of Arabidopsis plants [ 38 , 39 ]. The overexpression of PeGIF3 was established with a wild-type background. The single-insertion homozygous T3 lines of the PeGIF3 complement were carefully chosen and established in the atgif1 mutant background. Subcellular localization of PeGIF3 gene Transform the constructed 35S: PeGIF3 -YFP vector into Agrobacterium tumefaciens GV3101. Reconstitute the strain harboring the target plasmid(NLS-mcherry) in LB medium supplemented with appropriate antibiotics for overnight cultivation. Inoculate the bacterial solution obtained in the second step into fresh LB medium, simultaneously adding acetosyringone, and agitate until the bacteria reach an optical density (OD600) of 1.0-1.2. The supernatant should be discarded by centrifugation, and the bacteria should be resuspended in infection fluid (0.01M MES (pH = 5.6), 0.01M MgCl 2 ·6H 2 O and 50 µM acetosyringone) until the OD value reaches approximately 1.0. Allow it to remain undisturbed for a duration of 3 hours in a lightless environment. The target bacterial was combined with the NLS-mcherry in equal proportions, and tobacco were inoculated using a syringe. The treated plants were kept in darkness for 12 hours and subsequently incubated under normal conditions for 36 hours. The underlying epidermis of tobacco ( Nicotiana benthamiana ) was revealed in a dark environment and examined using a laser scanning confocal microscope (Nikon eclipse Ti2). The microscope was excited by a 488 nm laser and emitted signals were detected within the range of 500–550 nm. Measurement of leaf cell number The leaf cross-section chosen for anatomical analysis was carefully selected to encompass the widest point of the primary vein and subsequently fixed using FAA solution. The paraffin section method was employed to convert these into permanent film [ 40 ]. The samples were subsequently examined and imaged using a scanning electron microscope (OPLENIC CORP). Cells present in the pericycle to the leaf margin were enumerated. The statistical analyses were conducted using Graphpad Prism 9 software. The least significant difference test was employed to determine statistically significant differences between means at a significance level of p < 0.05 . Declarations Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 32160355). Author´s contributions YY, JS and CQ, carried out the experiment, collected and organized data and wrote the manuscript. PJ and ZW participated in designing the experiment and directed the study. ZL and ZW, reviewed the manuscript. YY and ZW, helped organize data. PJ, helped do the experiment. ZL and ZW, corresponding author, raised the hypothesis underlying this work, designed the experiment, and helped organize the manuscript structure. All authors read and approved the final manuscript. Funding This research was funded by Natural Science Foundation of China, grant number 32371838; the Biological Safety and Genetic Resources Management Project of the Science and Technology Development Center of the National Forestry and Grassland Administration, grant number KJZXSA202303 and Xinjiang Production and Construction Corps Regional Innovation Guidance Program project, grant number 2021BB010. Availability of data and materials RNA-seq data for P. euphratica 's heteromorphic leaves used in this study have been submitted to the NGDC (National Genomics Data Center, https://ngdc.cncb.ac.cn/) under the BioProject accession number PRJCA005959. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Bradshaw H, Ceulemans R, Davis J, Stettler R: Emerging model systems in plant biology: poplar (Populus) as a model forest tree . Journal of Plant Growth Regulation 2000, 19 (3):306-313. Brunner AM, Busov VB, Strauss SH: Poplar genome sequence: functional genomics in an ecologically dominant plant species . Trends in plant science 2004, 9 (1):49-56. Taylor G: Populus: Arabidopsis for forestry. Do we need a model tree? Annals of Botany 2002, 90 (6):681-689. Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S, Rombauts S, Salamov A: The genome of black cottonwood, Populus trichocarpa (Torr. & Gray) . Science 2006, 313 (5793):1596-1604. Qiu Q, Ma T, Hu Q, Liu B, Wu Y, Zhou H, Wang Q, Wang J, Liu J: Genome-scale transcriptome analysis of the desert poplar, Populus euphratica . Tree physiology 2011, 31 (4):452-461. Song Z, Ni X, Yao J, Wang F: Progress in studying heteromorphic leaves in Populus euphratica : leaf morphology, anatomical structure, development regulation and their ecological adaptation to arid environments . Plant signaling & behavior 2021, 16 (4):1870842. Liu Y, Li X, Chen G, Li M, Liu M, Liu D: Epidermal micromorphology and mesophyll structure of Populus euphratica heteromorphic leaves at different development stages . PLoS One 2015, 10 (9):e0137701. Hao J, Yue N, Zheng C: Analysis of changes in anatomical characteristics and physiologic features of heteromorphic leaves in a desert tree, Populus euphratica . Acta Physiologiae Plantarum 2017, 39 :1-11. Kim JH, Tsukaya H: Regulation of plant growth and development by the GROWTH-REGULATING FACTOR and GRF-INTERACTING FACTOR duo . Journal of experimental botany 2015, 66 (20):6093-6107. Kim JH, Kende H: A transcriptional coactivator, AtGIF1 , is involved in regulating leaf growth and morphology in Arabidopsis . Proc Natl Acad Sci U S A 2004, 101 (36):13374-13379. Horiguchi G, Kim GT, Tsukaya H: The transcription factor AtGRF5 and the transcription coactivator AN3 regulate cell proliferation in leaf primordia of Arabidopsis thaliana . The Plant journal : for cell and molecular biology 2005, 43 (1):68-78. Vercruyssen L, Verkest A, Gonzalez N, Heyndrickx KS, Eeckhout D, Han SK, Jégu T, Archacki R, Van Leene J, Andriankaja M et al : ANGUSTIFOLIA3 binds to SWI/SNF chromatin remodeling complexes to regulate transcription during Arabidopsis leaf development . The Plant cell 2014, 26 (1):210-229. Ercoli MF, Ferela A, Debernardi JM, Perrone AP, Rodriguez RE, Palatnik JF: GIF Transcriptional Coregulators Control Root Meristem Homeostasis . The Plant cell 2018, 30 (2):347-359. Li J, Pan W, Zhang S, Ma G, Li A, Zhang H, Liu L: A rapid and highly efficient sorghum transformation strategy using GRF4 ‐ GIF1/ternary vector system . The Plant Journal 2023. Lee BH, Ko JH, Lee S, Lee Y, Pak JH, Kim JH: The Arabidopsis GRF-INTERACTING FACTOR gene family performs an overlapping function in determining organ size as well as multiple developmental properties . Plant physiology 2009, 151 (2):655-668. Liu Y, Guo P, Wang J, Xu ZY: Growth ‐ regulating factors: conserved and divergent roles in plant growth and development and potential value for crop improvement . The Plant Journal 2023, 113 (6):1122-1145. Liang G, He H, Li Y, Wang F, Yu D: Molecular mechanism of microRNA396 mediating pistil development in Arabidopsis . Plant physiology 2014, 164 (1):249-258. Duan P, Ni S, Wang J, Zhang B, Xu R, Wang Y, Chen H, Zhu X, Li Y: Regulation of OsGRF4 by OsmiR396 controls grain size and yield in rice . Nature plants 2015, 2 :15203. Qin L, Chen H, Wu Q, Wang X: Identification and exploration of the GRF and GIF families in maize and foxtail millet . Physiology and Molecular Biology of Plants 2022, 28 (9):1717-1735. Zhang D, Sun W, Singh R, Zheng Y, Cao Z, Li M, Lunde C, Hake S, Zhang Z: GRF-interacting factor1 Regulates Shoot Architecture and Meristem Determinacy in Maize . The Plant cell 2018, 30 (2):360-374. Ai G, Zhang D, Huang R, Zhang S, Li W, Ahiakpa JK, Zhang J: Genome-Wide Identification and Molecular Characterization of the Growth-Regulating Factors-Interacting Factor Gene Family in Tomato . Genes 2020, 11 (12). Wu ZJ, Wang WL, Zhuang J: Developmental processes and responses to hormonal stimuli in tea plant ( Camellia sinensis ) leaves are controlled by GRF and GIF gene families . Functional & integrative genomics 2017, 17 (5):503-512. Zhang ZS, Zeng QY, Liu YJ: Frequent ploidy changes in Salicaceae indicates widespread sharing of the salicoid whole genome duplication by the relatives of Populus L. and Salix L . BMC plant biology 2021, 21 (1):535. Liu L, Xu W, Hu X, Liu H, Lin Y: W-box and G-box elements play important roles in early senescence of rice flag leaf . Scientific reports 2016, 6 :20881. Debernardi JM, Mecchia MA, Vercruyssen L, Smaczniak C, Kaufmann K, Inze D, Rodriguez RE, Palatnik JF: Post-transcriptional control of GRF transcription factors by microRNA miR396 and GIF co-activator affects leaf size and longevity . The Plant journal : for cell and molecular biology 2014, 79 (3):413-426. Yan S, Zou G, Li S, Wang H, Liu H, Zhai G, Guo P, Song H, Yan C, Tao Y: Seed size is determined by the combinations of the genes controlling different seed characteristics in rice . TAG Theoretical and applied genetics Theoretische und angewandte Genetik 2011, 123 (7):1173-1181. Li S, Gao F, Xie K, Zeng X, Cao Y, Zeng J, He Z, Ren Y, Li W, Deng Q et al : The OsmiR396c-OsGRF4-OsGIF1 regulatory module determines grain size and yield in rice . Plant biotechnology journal 2016, 14 (11):2134-2146. Xie Y, Skytting B, Nilsson G, Grimer RJ, Mangham CD, Fisher C, Shipley J, Bjerkehagen B, Myklebost O, Larsson O: The SYT-SSX1 fusion type of synovial sarcoma is associated with increased expression of cyclin A and D1. A link between t(X;18)(p11.2; q11.2) and the cell cycle machinery . Oncogene 2002, 21 (37):5791-5796. Nelissen H, Eeckhout D, Demuynck K, Persiau G, Walton A, van Bel M, Vervoort M, Candaele J, De Block J, Aesaert S et al : Dynamic Changes in ANGUSTIFOLIA3 Complex Composition Reveal a Growth Regulatory Mechanism in the Maize Leaf . The Plant cell 2015, 27 (6):1605-1619. Zhang Z, Chen Y, Zhang J, Ma X, Li Y, Li M, Wang D, Kang M, Wu H, Yang Y et al : Improved genome assembly provides new insights into genome evolution in a desert poplar ( Populus euphratica ) . Molecular ecology resources 2020, 20 (3). Xue L, Wu H, Chen Y, Li X, Hou J, Lu J, Wei S, Dai X, Olson MS, Liu J: Evidences for a role of two Y-specific genes in sex determination in Populus deltoides . Nature Communications 2020, 11 (1):5893. Zhou R, Macaya-Sanz D, Carlson CH, Schmutz J, Jenkins JW, Kudrna D, Sharma A, Sandor L, Shu S, Barry K et al : A willow sex chromosome reveals convergent evolution of complex palindromic repeats . Genome biology 2020, 21 (1):38. Dai X, Hu Q, Cai Q, Feng K, Ye N, Tuskan GA, Milne R, Chen Y, Wan Z, Wang Z et al : The willow genome and divergent evolution from poplar after the common genome duplication . Cell research 2014, 24 (10):1274-1277. Wu Z, Jiang Z, Li Z, Jiao P, Zhai J, Liu S, Han X, Zhang S, Sun J, Gai Z: Multi-omics analysis reveals spatiotemporal regulation and function of heteromorphic leaves in Populus . Plant physiology 2023, 192 (1):188-204. Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown . Nature protocols 2016, 11 (9):1650-1667. Kovaka S, Zimin AV, Pertea GM, Razaghi R, Salzberg SL, Pertea M: Transcriptome assembly from long-read RNA-seq alignments with StringTie2 . Genome biology 2019, 20 (1):1-13. Liu C, Hao J, Qiu M, Pan J, He Y: Genome-wide identification and expression analysis of the MYB transcription factor in Japanese plum ( Prunus salicina ) . Genomics 2020, 112 (6):4875-4886. Zhang X, Henriques R, Lin SS, Niu QW, Chua NH: Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method . Nature protocols 2006, 1 (2):641-646. Folkers U, Kirik V, Schöbinger U, Falk S, Krishnakumar S, Pollock MA, Oppenheimer DG, Day I, Reddy AS, Jürgens G et al : The cell morphogenesis gene ANGUSTIFOLIA encodes a CtBP/BARS-like protein and is involved in the control of the microtubule cytoskeleton . The EMBO journal 2002, 21 (6):1280-1288. Tsukaya H, Naito S, Rédei GP, Komeda Y: A new class of mutations in Arabidopsis thaliana, acaulis1, affecting the development of both inflorescences and leaves . Development (Cambridge, England) 1993, 118 (3):751-764. Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table.zip Supplementaryfile.zip Cite Share Download PDF Status: Published Journal Publication published 27 Mar, 2024 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 12 Feb, 2024 Reviews received at journal 11 Feb, 2024 Reviewers agreed at journal 06 Feb, 2024 Reviews received at journal 03 Feb, 2024 Reviewers agreed at journal 27 Jan, 2024 Reviewers agreed at journal 27 Jan, 2024 Reviewers invited by journal 27 Jan, 2024 Editor assigned by journal 27 Jan, 2024 Editor invited by journal 21 Jan, 2024 Submission checks completed at journal 21 Jan, 2024 First submitted to journal 20 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3881684","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":268380177,"identity":"2a7fb7bc-e871-418f-a905-1a501548bf4b","order_by":0,"name":"Yuqi Yang","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Yuqi","middleName":"","lastName":"Yang","suffix":""},{"id":268380178,"identity":"9fe84a02-ef60-4b2f-90b7-0dd204de4b67","order_by":1,"name":"Jianhao Sun","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Jianhao","middleName":"","lastName":"Sun","suffix":""},{"id":268380179,"identity":"7e4c5012-b93f-4cc7-a758-747b9b980ab7","order_by":2,"name":"Chen Qiu","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Qiu","suffix":""},{"id":268380180,"identity":"eb867603-796d-4d5e-9da0-109adef59442","order_by":3,"name":"Peipei Jiao","email":"","orcid":"","institution":"Tarim University","correspondingAuthor":false,"prefix":"","firstName":"Peipei","middleName":"","lastName":"Jiao","suffix":""},{"id":268380181,"identity":"2bf7ff54-eb68-45b4-a18b-7bb482e80e4e","order_by":4,"name":"Zhihua Wu","email":"","orcid":"","institution":"Zhejiang Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zhihua","middleName":"","lastName":"Wu","suffix":""},{"id":268380182,"identity":"b75e4abd-a063-44ae-8ed1-846aadc262e1","order_by":5,"name":"Zhijun Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIie3PwWrCQBCA4QmB3ctgrgmW+ApTAqVgHiZLYb2EIPgCSiFeAr2mb+EjKEPtxQfwmF48e0yhUDVF8OR4LLg/e5yPnQFwuf5hPR8UZAig9GzW7CmNRaLOJEDmx3psE5l071hUW9vHPRuZaL1rmgcuaJlTPyU/A80fC2GxEWXIE1puKMlJFYDWbgWyDo/ELFYVveSEEwjxSSBe+UcYiZ8pNFOZ+Koj76Uyr0B0C1EnMjJv6LNXUZYo6ZYg+NxF39XQlIOvedv+/MaB5vVV0uVVl/+K413tbWMul8t1px0A8HxEKOAyot8AAAAASUVORK5CYII=","orcid":"","institution":"Tarim University","correspondingAuthor":true,"prefix":"","firstName":"Zhijun","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-01-20 13:45:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3881684/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3881684/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-024-10221-5","type":"published","date":"2024-03-28T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50030368,"identity":"480756e4-38e0-485a-bd9c-bf96d1d687fe","added_by":"auto","created_at":"2024-01-23 12:03:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3258523,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromosomal distribution of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGIF\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes across six Salicaceae species.\u003c/strong\u003e (A) \u003cem\u003eP. euphratica\u003c/em\u003e; (B) \u003cem\u003eP. pruinose\u003c/em\u003e; (C) \u003cem\u003eP. deltoides\u003c/em\u003e; (D) \u003cem\u003eP. trichocarpa\u003c/em\u003e;\u003cem\u003e \u003c/em\u003e(E)\u003cem\u003e S. sinopurpurea\u003c/em\u003e;\u003cem\u003e \u003c/em\u003e(F) \u003cem\u003eS. suchowensis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3881684/v1/fdc788f2a400eb7156a8fb00.jpg"},{"id":50030363,"identity":"64f6cf2e-25e7-4f9e-8a3c-1a7eb71ae561","added_by":"auto","created_at":"2024-01-23 12:03:19","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1726141,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of conserved motifs and phylogenetic relationships among \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGIF \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenes across seven species. \u003c/strong\u003e(A) A neighbor-joining (NJ) phylogenetic tree between \u003cem\u003eP. euphratica\u003c/em\u003e and other six species followed by conserved motifs. The three groups were marked with different colors on tree branches. (B) The 30 \u003cem\u003eGIF\u003c/em\u003egenes in seven species have 5 conserved motifs.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3881684/v1/716cb0cc8f63c011eb1b5e82.jpg"},{"id":50030361,"identity":"0751ce47-6b32-4033-a9b3-fc43b98ba94a","added_by":"auto","created_at":"2024-01-23 12:03:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2906028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of collinearity among \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGIFs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e involving \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. euphratica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and six additional species. \u003c/strong\u003e(A) \u003cem\u003eP. euphratica\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e (B) \u003cem\u003eP. euphratica\u003c/em\u003e and \u003cem\u003eP. pruinose\u003c/em\u003e (C) \u003cem\u003eP. euphratica\u003c/em\u003e and \u003cem\u003eP. deltoides\u003c/em\u003e (D)\u003cem\u003e P. euphratica\u003c/em\u003e and\u003cem\u003e P. trichocarpa\u003c/em\u003e (E) \u003cem\u003eP. euphratica\u003c/em\u003e and \u003cem\u003eS. sinopurpurea \u003c/em\u003e(F) \u003cem\u003eP. euphratica\u003c/em\u003e and \u003cem\u003eS. suchowensis\u003c/em\u003e. The presence of gray lines in the background denotes collinear blocks within \u003cem\u003eP. euphratica\u003c/em\u003e and other plant genomes, while the red lines emphasize collinear \u003cem\u003eGIF\u003c/em\u003e pairs.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3881684/v1/ab75e14989005989889045dd.jpg"},{"id":50030581,"identity":"a78d4610-78c8-4a75-99f2-12878495c605","added_by":"auto","created_at":"2024-01-23 12:11:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4211463,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCis-element analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGIF\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epromoters in six Salicaceae\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e species\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(A) The numbers and gradient red colors serve as indicators of the abundance of cis-acting elements present in each gene. (B) Color-coded histograms depict the distribution of cis-acting elements in each gene, categorized into three distinct groups. (C) Pie charts depicting the distribution of distinct cis-acting elements within each category.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3881684/v1/f3ca95653c5906fbfc0f31d8.jpg"},{"id":50030582,"identity":"130fc351-a533-4071-896d-d803bcc8abf0","added_by":"auto","created_at":"2024-01-23 12:11:19","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1094286,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePeGIFs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene expression patterns in heteromorphic leaves. (A)\u003c/strong\u003e Expression patterns of \u003cem\u003ePeGIFs \u003c/em\u003eacross three stages in four heteromorphic leaves; \u003cstrong\u003e(B) \u003c/strong\u003eqRT−PCR data on the expression patterns of \u003cem\u003ePeGIF3 \u003c/em\u003efor heteromorphic leaves in P1 stage; \u003cstrong\u003e(C) \u003c/strong\u003eqRT−PCR data on the expression patterns of other \u003cem\u003ePeGIFs \u003c/em\u003efor heteromorphic leaves in P1 stage.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3881684/v1/8b94fb75502785d5be694129.jpg"},{"id":50030365,"identity":"a75793df-af8e-4262-b6e3-92bce48f0ac3","added_by":"auto","created_at":"2024-01-23 12:03:19","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1151395,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFluorescent images of 35S:\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePeGIF3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-YFP.\u003c/strong\u003e \u003cstrong\u003eBar = 50 mm\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3881684/v1/79f6c6eb47a8d51eb6de9c8e.jpg"},{"id":50030364,"identity":"b82f144c-106f-4f58-b686-2d2acdec30a8","added_by":"auto","created_at":"2024-01-23 12:03:19","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1234067,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePeGIF3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene regulates leaf cell morphology. \u003c/strong\u003e(A) The cross-sectional anatomical structure of the fifth leaf rosette in each plant. (B) The overall morphological characteristics of various plant species. (C) Relevance analysis of cellular quantities across diverse plant species (****, \u003cem\u003eP\u003c/em\u003e ≤ 0.0001；ns, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3881684/v1/5ecf464c11bd5df90e457e5c.jpg"},{"id":53733195,"identity":"be60667c-de53-4afe-8d12-9e333ccd3e21","added_by":"auto","created_at":"2024-03-29 13:50:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2413950,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3881684/v1/bb6c9052-b680-46df-865d-da4b726eee31.pdf"},{"id":50030580,"identity":"534998be-fa50-413e-8e71-698498794c91","added_by":"auto","created_at":"2024-01-23 12:11:19","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18130,"visible":true,"origin":"","legend":"","description":"","filename":"Table.zip","url":"https://assets-eu.researchsquare.com/files/rs-3881684/v1/991a2764deff4557afc10772.zip"},{"id":50030367,"identity":"f328ff89-0475-49ba-bada-ed6e560d00f5","added_by":"auto","created_at":"2024-01-23 12:03:19","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":376525,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.zip","url":"https://assets-eu.researchsquare.com/files/rs-3881684/v1/4c3065d1b00716d6e5cf6481.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative genomic analysis of the Growth Regulating Factors-Interacting Factors (GIFs) in six Salicaceae species and functional analysis of PeGIF3 reveals their regulatory role in Populus heteromorphic leaves","fulltext":[{"header":"Background","content":"\u003cp\u003eSalicaceae species are commonly chosen as exemplary forest trees in various research studies due to their ease of vitro regeneration, rapid vegetative reproduction, and significant ecological and economic importance across the northern hemisphere [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Especially, \u003cem\u003ePopulus euphratica\u003c/em\u003e characterized by its extraordinary tolerance to both drought and salinity, serves as a pivotal species in desert oases and stands out as an exceptional relic plant thriving in extremely arid environments [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eP. euphratica\u003c/em\u003e exhibits a distinct pattern of leaf heteroblasty, with adult trees displaying a sequential production of linear (Li, leaf index\u0026thinsp;\u0026ge;\u0026thinsp;4), lanceolate (La, 2\u0026thinsp;\u0026le;\u0026thinsp;leaf index\u0026thinsp;\u0026lt;\u0026thinsp;4), ovate (Ov, 1\u0026thinsp;\u0026le;\u0026thinsp;leaf index\u0026thinsp;\u0026lt;\u0026thinsp;2), and broad ovate (Bo, leaf index\u0026thinsp;\u0026lt;\u0026thinsp;1) leaves as they ascend from the lower to upper canopies, accompanied by a gradual increase in leaf width and area. Previous research has indicated that broad Ov and Bo leaves in \u003cem\u003eP. euphratica\u003c/em\u003e exhibit greater resilience to drought compared to narrow Li and La leaves, as evidenced by their thicker palisade tissue and enhanced photosynthetic activity. In addition, there was a significant and positive association observed between leaf area and the content of proline [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. So, it is believed that the presence of heteromorphic leaves in \u003cem\u003eP. euphratica\u003c/em\u003e suggests its capacity to adjust to the dry desert environment. Hence, it is crucial to identify and characterize the genes involving in the heteromorphic leaf development of \u003cem\u003eP. euphratica\u003c/em\u003e to reveal the functional divergence and adaptive evolution of heteromorphic leaves.\u003c/p\u003e \u003cp\u003e \u003cem\u003eGrowth-Regulating Factors-Interacting Factors\u003c/em\u003e (\u003cem\u003eGIFs\u003c/em\u003e) represent a class of transcriptional co-activators that collaborate with \u003cem\u003eGrowth-Regulating Factors\u003c/em\u003e (\u003cem\u003eGRFs\u003c/em\u003e). Typically, \u003cem\u003eGIFs\u003c/em\u003e can interact with \u003cem\u003eGRFs\u003c/em\u003e, forming a plant-specific transcriptional complex [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The \u003cem\u003eGIF\u003c/em\u003e gene family was initially discovered in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e in 2004[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. \u003cem\u003eGIF\u003c/em\u003e genes have been intricately linked with plant growth and development [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In a recent study, it was revealed that \u003cem\u003eGIFs\u003c/em\u003e play a vital role in maintaining the precise expression patterns of key developmental factors [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cem\u003eGIF\u003c/em\u003e transcriptional coregulators assume the responsibility of regulating the quiescent center organization and the meristem size in \u003cem\u003eA. thaliana\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. \u003cem\u003eGIF2\u003c/em\u003e and \u003cem\u003eGIF3\u003c/em\u003e are two additional proteins instrumental in cell proliferation and the development of lateral organs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The \u003cem\u003egif2\u003c/em\u003e and \u003cem\u003egif3\u003c/em\u003e mutations have been associated with the production of smaller lateral organs in comparison to wild-type plant species in \u003cem\u003eA. thaliana\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Single \u003cem\u003egif\u003c/em\u003e mutant lines in \u003cem\u003eA. thaliana\u003c/em\u003e presented a phenotype akin to the control, but the \u003cem\u003egif\u003c/em\u003e triple mutant \u003cem\u003egif1/gif2/gif3\u003c/em\u003e exhibited an aberrant pistil [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBeyond their roles in model plants, \u003cem\u003eGIFs\u003c/em\u003e exert considerable influence in rice [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], maize[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], tomato[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], tea[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Although \u003cem\u003eGIFs\u003c/em\u003e have been investigated in certain plant species, further exploration of this gene family is warranted in Salicaceae. Recent advancements in genomics of Salicaceae species offer an opportunity to characterize the \u003cem\u003eGIF\u003c/em\u003e gene family. In this research, we conducted the genome-wide analysis of the \u003cem\u003eGIF\u003c/em\u003e gene family, and characterize their structures, conserved motifs, cis-elements, and expression patterns, as well as the comparative genomics analysis of \u003cem\u003eGIF\u003c/em\u003e gene family across six different Salicaceae species. We further revealed the role of \u003cem\u003ePeGIF3\u003c/em\u003e in heteromorphic leaf development in \u003cem\u003eP. euphratica\u003c/em\u003e through heterologous expression in \u003cem\u003eA. thaliana\u003c/em\u003e. These results obtained from this research will offer crucial and precious data for forthcoming investigations concerning the functional characterization of the \u003cem\u003eGIF\u003c/em\u003e gene family in Salicaceae species.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentification and characterization of\u003c/b\u003e \u003cb\u003eGIF\u003c/b\u003e\u003cb\u003es in six Salicaceae species\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 4, 4, 6, 4, 4, and 5 \u003cem\u003eGIF\u003c/em\u003e genes were identified in \u003cem\u003eP. euphratica\u003c/em\u003e, \u003cem\u003eP. pruinose\u003c/em\u003e, \u003cem\u003eP. deltoides\u003c/em\u003e, \u003cem\u003eP. trichocarpa\u003c/em\u003e, \u003cem\u003eS. sinopurpurea\u003c/em\u003e, and \u003cem\u003eS. suchowensis\u003c/em\u003e, respectively. Then, according to the location on chromosomes, the \u003cem\u003eGIF\u003c/em\u003e members within the Salicaceae species were assigned names as follows: \u003cem\u003ePeGIF1\u003c/em\u003e to \u003cem\u003ePeGIF4\u003c/em\u003e; \u003cem\u003ePpGIF1\u003c/em\u003e to \u003cem\u003ePpGIF4\u003c/em\u003e; \u003cem\u003ePdGIF1\u003c/em\u003e to \u003cem\u003ePdGIF6\u003c/em\u003e; \u003cem\u003ePtGIF1\u003c/em\u003e to \u003cem\u003ePtGIF4\u003c/em\u003e; \u003cem\u003eSPUGIF1\u003c/em\u003e to \u003cem\u003eSPUGIF4\u003c/em\u003e; \u003cem\u003eSSUGIF1\u003c/em\u003e to \u003cem\u003eSSUGIF5\u003c/em\u003e. Almost all of \u003cem\u003eGIF\u003c/em\u003e genes were located on single chromosome in six Salicaceae species (Fig.\u0026nbsp;1). These results suggest that the number of \u003cem\u003eGIF\u003c/em\u003e gene family is relatively conserved without dramatic expansion or loss in Salicaceae. Subsequently, we conducted a comprehensive analysis on the characteristics of \u003cem\u003eGIF\u003c/em\u003e genes and their encoded proteins (Table\u0026nbsp;1). The analysis of protein sequences revealed that the GIF proteins have the potential to encode amino acids ranging from 79 to 223, with a molecular weight (MW) varying between 8930.16 and 22222.92 kDa. Additionally, their isoelectric point was observed to fall within the range of 4.71 to 5.93.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable\u0026nbsp;1 Characteristics of the putative\u003c/b\u003e \u003cb\u003eGIF\u003c/b\u003e \u003cb\u003egenes.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 1. Chromosomal distribution of\u003c/b\u003e \u003cb\u003eGIF\u003c/b\u003e \u003cb\u003egenes across six Salicaceae species.\u003c/b\u003e (A) \u003cem\u003eP. euphratica\u003c/em\u003e; (B) \u003cem\u003eP. pruinose\u003c/em\u003e; (C) \u003cem\u003eP. deltoides\u003c/em\u003e; (D) \u003cem\u003eP. trichocarpa\u003c/em\u003e; (E) \u003cem\u003eS. sinopurpurea\u003c/em\u003e; (F) \u003cem\u003eS. suchowensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of phylogenetic and conserved motifs in multi-species of\u003c/b\u003e \u003cb\u003eGIFs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe evolutionary relationships and potential functions of 30 \u003cem\u003eGIFs\u003c/em\u003e from six Salicaceae and one Brassicaceae species were investigated by constructing a phylogenetic tree. The analysis confirmed the classification of \u003cem\u003eGIF\u003c/em\u003e genes into three subfamilies, denoted as Group I, II, and III. Specifically, Group I encompassed 13 genes (2 \u003cem\u003eATGIFs\u003c/em\u003e, 2 \u003cem\u003ePeGIFs\u003c/em\u003e, 2 \u003cem\u003ePpGIF\u003c/em\u003e, \u003cem\u003e2 PdGIFs\u003c/em\u003e, 2 \u003cem\u003ePtGIFs\u003c/em\u003e, 2 \u003cem\u003eSPUGIFs\u003c/em\u003e, and \u003cem\u003eSSUGIF3\u003c/em\u003e), Group II comprised 12 genes (\u003cem\u003eATGIF1\u003c/em\u003e, \u003cem\u003ePeGIF3\u003c/em\u003e, \u003cem\u003ePpGIF2\u003c/em\u003e, 2 \u003cem\u003ePtGIFs\u003c/em\u003e, 2 \u003cem\u003ePdGIFs\u003c/em\u003e, 2 \u003cem\u003eSPUGIFs\u003c/em\u003e, and 3 \u003cem\u003eSSUGIFs\u003c/em\u003e), while the Group III contained five genes (2 \u003cem\u003ePdGIFs\u003c/em\u003e, \u003cem\u003ePpGIF4\u003c/em\u003e, \u003cem\u003ePeGIF2\u003c/em\u003e, and \u003cem\u003eSSUGIF1\u003c/em\u003e) uniquely belonged to the Salicaceae (Fig.\u0026nbsp;2A). The unique GIFs from Group III indicated they occurred after the divergence between Arabidopsis and Salicaceae. Then, MEME software was employed to predict conserved motifs in these \u003cem\u003eGIF\u003c/em\u003e genes. Five motifs were identified, with motif 1 representing the conserved SSXT(SNH) domain located in the N-terminal region, common among most \u003cem\u003eGIF\u003c/em\u003e genes. Notably, motif 3 and motif 4 were present in most genes, while motif 2 exclusively appeared in Group I and Group II genes. Additionally, motif 5 was primarily found in Group II members, hinting at its uniqueness to this group. Interestingly, motifs 2 and 5 at C-terminus were presented in \u003cem\u003eGIF\u003c/em\u003e members of only Salicaceae species, possibly indicating their unique roles. Therefore, besides the expanded gene number of \u003cem\u003eGIFs\u003c/em\u003e in Salicaceae, the variations of GIF domain numbers may also contribute to the novelty of \u003cem\u003eGIFs\u003c/em\u003e in Salicaceae.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2. Analysis of conserved motifs and phylogenetic relationships among\u003c/b\u003e \u003cb\u003eGIF\u003c/b\u003e \u003cb\u003egenes across seven species.\u003c/b\u003e (A) A neighbor-joining (NJ) phylogenetic tree between \u003cem\u003eP. euphratica\u003c/em\u003e and other six species followed by conserved motifs. The three groups were marked with different colors on tree branches. (B) The 30 \u003cem\u003eGIF\u003c/em\u003e genes in seven species have 5 conserved motifs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCollinearity analysis of multi-species\u003c/b\u003e \u003cb\u003eGIFs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further investigate the evolutionary processes of \u003cem\u003ePeGIFs\u003c/em\u003e in \u003cem\u003ePopulus\u003c/em\u003e, we performed the collinear analysis of \u003cem\u003eGIFs\u003c/em\u003e between \u003cem\u003eP. euphratica\u003c/em\u003e and other 6 species (\u003cem\u003eA. thaliana, P. pruinose, P. deltoides\u003c/em\u003e, \u003cem\u003eP. trichocarpa\u003c/em\u003e, \u003cem\u003eS. sinopurpurea\u003c/em\u003e and \u003cem\u003eS. suchowensis\u003c/em\u003e). The number of \u003cem\u003eGIF\u003c/em\u003e collinear fragements between \u003cem\u003eP. euphratica\u003c/em\u003e and other species were as follows: 8 pairs with \u003cem\u003eP. pruinose\u003c/em\u003e and \u003cem\u003eS. suchowensis\u003c/em\u003e, 12 pairs with \u003cem\u003eP. deltoides\u003c/em\u003e and \u003cem\u003eP. trichocarpa\u003c/em\u003e, and 5 pairs with \u003cem\u003eS. sinopurpurea\u003c/em\u003e, respectively. This result revealed that the collinearity of \u003cem\u003eGIFs\u003c/em\u003e within poplar species was more conservative than that between \u003cem\u003eP. euphratica\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e (5 collinear fragements). Notably, 50 pairs of \u003cem\u003ePeGIF\u003c/em\u003e genes exhibited collinear relationships between \u003cem\u003eP. euphratica\u003c/em\u003e and the other five Salicaceae species, indicating that these \u003cem\u003eGIF\u003c/em\u003e included collinear fragments likely predated the ancestral divergence (Fig.\u0026nbsp;3). The retention of \u003cem\u003eGIF\u003c/em\u003e included collinear fragments possibly resulted from the whole genome duplication event occurred in Salicaceae [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 3. Analysis of collinearity among\u003c/b\u003e \u003cb\u003eGIFs\u003c/b\u003e \u003cb\u003einvolving\u003c/b\u003e \u003cb\u003eP. euphratica\u003c/b\u003e \u003cb\u003eand six additional species.\u003c/b\u003e (A) \u003cem\u003eP. euphratica\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e (B) \u003cem\u003eP. euphratica\u003c/em\u003e and \u003cem\u003eP. pruinose\u003c/em\u003e (C) \u003cem\u003eP. euphratica\u003c/em\u003e and \u003cem\u003eP. deltoides\u003c/em\u003e (D) \u003cem\u003eP. euphratica\u003c/em\u003e and \u003cem\u003eP. trichocarpa\u003c/em\u003e (E) \u003cem\u003eP. euphratica\u003c/em\u003e and \u003cem\u003eS. sinopurpurea\u003c/em\u003e (F) \u003cem\u003eP. euphratica\u003c/em\u003e and \u003cem\u003eS. suchowensis\u003c/em\u003e. The presence of gray lines in the background denotes collinear blocks within \u003cem\u003eP. euphratica\u003c/em\u003e and other plant genomes, while the red lines emphasize collinear \u003cem\u003eGIF\u003c/em\u003e pairs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of cis-regulatory elements of\u003c/b\u003e \u003cb\u003eGIF\u003c/b\u003e \u003cb\u003eGenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo analyze the possible factors influencing the expression patterns of \u003cem\u003eGIF\u003c/em\u003e gene family members, we used the PlantCARE online website to predict cis-acting elements. As results, the promoters of almost all \u003cem\u003eGIF\u003c/em\u003es contained various cis-acting elements associated with plant growth and development, phytohormone responses and stress responses. For instance, elements related to plant development, such as CAT-box, TGA-box, AAGAA-motif, GCN4-motif, as-1, Box4 and G-box, etc (Fig.\u0026nbsp;4A). Among these elements, all genes have Box4 elements, which are part of the conserved DNA module involved in light response. In addition, G-box were important for early senescence of rice flag leaves [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Notably, auxin-related elements (TGA-box) were present in most members of each species, suggesting a role for \u003cem\u003eGIF\u003c/em\u003e genes in regulating \u003cem\u003ePopulus\u003c/em\u003e growth and development. In addition to these elements, there were other elements related to growth and development in some \u003cem\u003eGIF\u003c/em\u003e genes, including the CAT-box related to meristem expression, GCN4-motif related to endosperm expression. Meanwhile, stress-related elements encompass anaerobic and anoxic conditions, defense mechanisms against stressors, drought tolerance, low temperature adaptation, and wound response. These elements are regulated by specific cis-acting motifs including antioxidant response element (ARE), GC-rich motif (GC-motif), TC-rich repeats (TCRRs), MYB binding site (MBS), low temperature-responsive element (LTR), and wound-induced promoter motif (WUN-motif). Interestingly, the presence of MYB elements associated with drought stress response was observed in all \u003cem\u003eGIFs\u003c/em\u003e, indicating their crucial role in mediating the response to drought-induced stress. Notably, among the hormone-responsive elements, prominent ones included abscisic acid (ABRE), auxin (TGA-element), gibberellin (TATC-box, P-box, GARE-motif), MeJA (TGACG-motif, CGTCA-motif), and salicylic acid (TCA-element). Also, cis-acting elements related to MeJA were the most prevalent among hormone-responsive elements. This analysis suggests that \u003cem\u003eGIF\u003c/em\u003e genes participate in diverse growth and developmental processes possibly mediated by hormone signal transduction or environmental stimulus (Fig.\u0026nbsp;4B).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 4. Cis-element analysis of\u003c/b\u003e \u003cb\u003eGIF\u003c/b\u003e \u003cb\u003epromoters in six Salicaceae\u003c/b\u003e \u003cb\u003especies\u003c/b\u003e. (A) The numbers and gradient red colors serve as indicators of the abundance of cis-acting elements present in each gene. (B) Color-coded histograms depict the distribution of cis-acting elements in each gene, categorized into three distinct groups. (C) Pie charts depicting the distribution of distinct cis-acting elements within each category.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression of\u003c/b\u003e \u003cb\u003ePeGIFs\u003c/b\u003e \u003cb\u003ein heteromorphic leaves from juvenile to adult\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the transcriptional regulation of \u003cem\u003ePeGIFs\u003c/em\u003e underlying the developmental and functional differentiation processes of heteromorphic leaves, we assessed the expression level on four types of heteromorphic leaves (Li, La, Ov, and Bo) at three different developmental stages (P1, P2, and P3). \u003cem\u003ePeuTF02G01597\u003c/em\u003e (\u003cem\u003ePeGIF1\u003c/em\u003e), \u003cem\u003ePeuTF12G00083\u003c/em\u003e (\u003cem\u003ePeGIF2\u003c/em\u003e), and \u003cem\u003ePeuTF14G00928\u003c/em\u003e (\u003cem\u003ePeGIF4\u003c/em\u003e) exhibited low expression levels. In contrast, \u003cem\u003ePeuTF13G00452(PeGIF3)\u003c/em\u003e, which is homologous to \u003cem\u003eATGIF1\u003c/em\u003e in \u003cem\u003eA. thaliana\u003c/em\u003e and plays a crucial role in leaf development, was specifically upregulated in Ov and Bo leaves at the early stage of leaf development (Fig.\u0026nbsp;5A). Moreover, the expression level of \u003cem\u003ePeGIF3\u003c/em\u003e significantly decreased during later stages of leaf growth (P2-P3), suggesting its potential involvement in promoting broad-leaf expansion during early morphogenesis. Collectively, our results suggest that \u003cem\u003ePeGIF3\u003c/em\u003e plays a dynamic role in regulating the development of broad leaves (Ov and Bo) in \u003cem\u003eP. euphratica\u003c/em\u003e, qRT-PCR analysis was further adopted for confirmation of \u003cem\u003ePeGIFs\u003c/em\u003e\u0026rsquo; expression in heteromorphic leaves at the P1 leaf stage (Fig.\u0026nbsp;5B\u0026thinsp;~\u0026thinsp;5C). All the \u003cem\u003ePeGIFs\u003c/em\u003e evaluated by qRT-PCR showed the similar expression pattern from RNA-seq result. Collectively, \u003cem\u003ePeGIF3\u003c/em\u003e also encompasses multiple growth-related elements (AAGAA-motif and as-1), particularly auxin-related elements (TGA-box). These results indicated up-regulation of \u003cem\u003ePeGIF3\u003c/em\u003e would regulate the occurrence of broad heteromorphic leaves in \u003cem\u003eP. euphratica\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 5. The\u003c/b\u003e \u003cb\u003ePeGIFs\u003c/b\u003e \u003cb\u003egene expression patterns in heteromorphic leaves. (A)\u003c/b\u003e Expression patterns of \u003cem\u003ePeGIFs\u003c/em\u003e across three stages in four heteromorphic leaves; \u003cb\u003e(B)\u003c/b\u003e qRT\u0026thinsp;\u0026minus;\u0026thinsp;PCR data on the expression patterns of \u003cem\u003ePeGIF3\u003c/em\u003e for heteromorphic leaves in P1 stage; \u003cb\u003e(C)\u003c/b\u003e qRT\u0026thinsp;\u0026minus;\u0026thinsp;PCR data on the expression patterns of other \u003cem\u003ePeGIFs\u003c/em\u003e for heteromorphic leaves in P1 stage.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSubcellular localization of\u003c/b\u003e \u003cb\u003ePeGIF3\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the subcellular distribution of the PeGIF3 protein in plant cells, the GV3101 \u003cem\u003eAgrobacterium\u003c/em\u003e strain carrying the 35S:\u003cem\u003ePeGIF3\u003c/em\u003e-YFP construct was introduced into tobacco, and the subcellular localization of \u003cem\u003ePeGIF3\u003c/em\u003e was visualized using a confocal laser scanning microscope. The fluorescence signal of 35S:\u003cem\u003ePeGIF3\u003c/em\u003e-YFP coincides with the nuclear localization signal of NLS-mCherry (Fig.\u0026nbsp;6). The subcellular localization of PeGIF3 was observed to be predominantly in the nucleus.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 6. Fluorescent images of 35S\u003c/b\u003e:\u003cb\u003ePeGIF3\u003c/b\u003e\u003cb\u003e-YFP. Bar =\u0026thinsp;50 mm\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe function of\u003c/b\u003e \u003cb\u003ePeGIF3\u003c/b\u003e \u003cb\u003einvolving leaf cell morphology\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the cellular underpinnings of transgenic plant phenotypes, we conducted an analysis on the fifth leaves derived from wild-type plants, \u003cem\u003eatgif1\u003c/em\u003e mutant plants, transgenic wild-type plants overexpressing the \u003cem\u003ePeGIF3\u003c/em\u003e gene, as well as the complementation of \u003cem\u003ePeGIF3\u003c/em\u003e in \u003cem\u003eatgif1\u003c/em\u003e mutant plants (Table\u0026nbsp;2). Twenty specimens of each plant were selected for sampling. The \u003cem\u003eatgif1\u003c/em\u003e mutant exhibited a more pronounced leaf size defect and a greater reduction in cell number in leaves in this study. To the contrary, the number of cells was significantly higher in overexpressed transgenic plants compared to wild-type plants, indicating a marked increase in cell abundance. The phenotypes of transgenic \u003cem\u003eatgif1\u003c/em\u003e plants expressing the \u003cem\u003ePeGIF3\u003c/em\u003e gene were comparable to those of the wild type (Fig.\u0026nbsp;7).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable\u0026nbsp;2. Transgenic plants of this work.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 7. The\u003c/b\u003e \u003cb\u003ePeGIF3\u003c/b\u003e \u003cb\u003egene regulates leaf cell morphology.\u003c/b\u003e (A) The cross-sectional anatomical structure of the fifth leaf rosette in each plant. (B) The overall morphological characteristics of various plant species. (C) Relevance analysis of cellular quantities across diverse plant species (****, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001;ns, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHeteromorphic leaves in \u003cem\u003eP. euphratica\u003c/em\u003e exhibit functional divergence at both physiological and cytological levels. GIF proteins, recognized as key players in leaf development, positively regulate leaf size. Previous investigations have predominantly centered on \u003cem\u003eGIF\u003c/em\u003e genes involvement in plant growth and development. This includes their role in maintaining the homeostasis of leaf, seed, and root meristems in \u003cem\u003eA. thaliana\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. \u003cem\u003eGIF\u003c/em\u003e genes have also been associated with modulating tissue and organ size in rice [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In maize, they have been implicated in regulating shoot architecture and meristem determinacy [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, their presence and characteristics in Salicaceae remain unexplored. Recent releases of high-quality genomes for Salicaceae species, including \u003cem\u003eP. euphratica\u003c/em\u003e, \u003cem\u003eP. pruinose, P. deltoides\u003c/em\u003e, \u003cem\u003eP. trichocarpa\u003c/em\u003e, \u003cem\u003eS. sinopurpurea\u003c/em\u003e, and \u003cem\u003eS. suchowensis\u003c/em\u003e, have opened new avenues for studying the \u003cem\u003eGIF\u003c/em\u003e family in Salicaceae. In this study, we identified a total of 27 members belonging to the \u003cem\u003eGIF\u003c/em\u003e family within six Salicaceae species, and delve into their structures and phylogenetic relationships within these species.\u003c/p\u003e \u003cp\u003eTo confirm the evolutionary relationships between \u003cem\u003eGIFs\u003c/em\u003e, the 27 \u003cem\u003eGIF\u003c/em\u003e members were classified into 3 subfamilies based on domain analysis and phylogenetic tree. The presence of highly conserved domains in the GIF protein within the same group suggests a potential similarity in function. Previously, the \u003cem\u003eATGIF1\u003c/em\u003e transcription coactivator gene was previously characterized as a positive regulator of cell proliferation in lateral organs, such as leaves and flowers, of \u003cem\u003eA. thaliana\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. We hypothesized that members located in the same subfamily as \u003cem\u003eATGIF1\u003c/em\u003e also function to regulate plant growth.\u003c/p\u003e \u003cp\u003eThe presence of the highly-conserved SSXT motif was detected in all members comprising the \u003cem\u003eGIF\u003c/em\u003e gene family. The findings are in line with prior research [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The N-terminal region of GIF proteins shares similarity with the SNH domain discovered in SYNOVIAL TRANSLOCATION (SYT) in humans, which interacts with BRAHMA (BRM) and BRAHMA RELATED GENE1 (BRG1), two ATPases involved in SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin remodeling processes in human cells. Based on the similarity in sequence, it is probable that \u003cem\u003eGIF\u003c/em\u003e transcriptional coactivators facilitate transcription through their interaction with SWI/SNF chromatin remodelers [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Additional motifs were also found in some \u003cem\u003eGIF\u003c/em\u003e members, implying that these members might have undergone functional divergence or acquired novel functions throughout the course of plant evolution.\u003c/p\u003e \u003cp\u003eOur promoter analysis unveiled a significant number of phytohormone-responsive elements (ABRE, CGTCA, and TGACG), light-responsive elements (G-box, GT1, and Box 4), and stress-responsive elements (ARE, MYB, and MYC) in the promoter regions of these \u003cem\u003eGIF\u003c/em\u003e genes. Notably, these \u003cem\u003eGIF\u003c/em\u003e gene members have been linked to plant development, a finding consistent with prior research in \u003cem\u003eA. thaliana\u003c/em\u003e [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnalysis of gene expression patterns in heteromorphic leaves led to the identification of differentially regulated genes specific to \u003cem\u003eP. euphratica\u003c/em\u003e. The expression level of \u003cem\u003ePeGIF3\u003c/em\u003e was found to be significantly higher and exhibited a notable disparity between heteromorphic leaves at the P1 age, which is consistent with the qRT-PCR results. Therefore, we hypothesize that \u003cem\u003ePeGIF3\u003c/em\u003e may be closely regulated in increasing the size of broad leaves early during leaf morphogenesis in \u003cem\u003eP. euphratica\u003c/em\u003e. By examining transgenic plants, we observed a significant difference in the number of leaf cells between the overexpressed plants and the wild type. The subsequent observation revealed that \u003cem\u003ePeGIF3\u003c/em\u003e was predominantly observed in the nucleus. It consistent with the conserved motifs analysis of \u003cem\u003eGIFs\u003c/em\u003e in multi-species in this study. Therefore, we hypothesized that \u003cem\u003ePeGIF3\u003c/em\u003e enhances leaf cell proliferation by modulating transcriptional processes, thereby resulting in the expansion of the central-lateral region of the leaf.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we firstly identified 27 \u003cem\u003eGIF\u003c/em\u003e genes in six Salicaceae species and characterize their structures, phylogenetic relationships, conserved motifs and collinearity across Salicaceae species with Arabidopsis as an outgroup. Detailed cis-elements analysis showed the Salicaceae \u003cem\u003eGIFs\u003c/em\u003e involved in multiple developmental processes and were regulated by diverse factors, such as phytohormones signals and environmental stimulus. Importantly, only \u003cem\u003ePeGIF3\u003c/em\u003e showed the gradual upregulation along with the development of heteromorphic leaves of Li, La, Ov and Bo, successively. The essential involvement of \u003cem\u003ePeGIF3\u003c/em\u003e in \u003cem\u003eP. euphratica\u003c/em\u003e leaf development has been elucidated using RNA-Seq data and qRT-PCR. Further overexpression of \u003cem\u003ePeGIF3\u003c/em\u003e in \u003cem\u003eatgif1\u003c/em\u003e mutant and wild-type of Arabidopsis results in enhanced leaf expansion along the medial-lateral and an increased cell population. Our findings provide a strong foundation for further functional investigations into \u003cem\u003eGIF\u003c/em\u003e genes in Salicaceae species and also promote the study of leaf morphological variation among Salicaceae species.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eIdentification characterization of\u003c/b\u003e \u003cb\u003eGRF-Interacting Factor\u003c/b\u003e \u003cb\u003ehomologs in Salicaceae\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ePeGIFs\u003c/em\u003e were identified based on our \u003cem\u003eP. euphratica\u003c/em\u003e genome data [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The hidden Markov model (HMM) profiles for the GIF domain SSXT (PF05030) were acquired from the Pfam protein family database, accessible at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org\u003c/span\u003e\u003cspan address=\"http://pfam.xfam.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. HMMER 3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://hmmer.org/\u003c/span\u003e\u003cspan address=\"http://hmmer.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed to conduct a search for potential \u003cem\u003eGIF\u003c/em\u003e genes in the six Salicaceae species. The superfluous candidate genes were excluded, and the remaining genes underwent additional validation using SMART (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.emblheidelberg.de/\u003c/span\u003e\u003cspan address=\"http://smart.emblheidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The protein physicochemical properties of GIF proteins, such as the amino acid count, molecular weight (MW), and theoretical isoelectric point (pI), were determined using the ProtParam tool available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"http://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. In addition, the \u003cem\u003eGIFs\u003c/em\u003e from five other Salicaceae species were identified based on the genome data of \u003cem\u003ePopulus pruinose\u003c/em\u003e (National Center for Biotechnology Information(NCBI) with the BioProject accession number PRJNA863418), \u003cem\u003ePopulus deltoides\u003c/em\u003e (WV94_445) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], \u003cem\u003ePopulus trichocarpa\u003c/em\u003e (V3.1) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], \u003cem\u003eSalix sinopurpurea\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and \u003cem\u003eSalix suchowensis\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The chromosomal location of \u003cem\u003eGIFs\u003c/em\u003e was obtained from the genome annotation files, and the chromosome physical location of the \u003cem\u003eGIF\u003c/em\u003e genes was displayed by MapChart V2.32 software.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic relationship consensus sequence analysis of multi-species\u003c/b\u003e \u003cb\u003eGIFs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the evolutionary relationship among \u003cem\u003eGIF\u003c/em\u003e genes, a phylogenetic tree was constructed using the amino acid sequences that encode \u003cem\u003eGIF\u003c/em\u003e genes from \u003cem\u003eP. euphratica\u003c/em\u003e and various other species. The SMART website was utilized to extract the domain coordinates from the GIF protein sequence of \u003cem\u003eP. euphratica\u003c/em\u003e and various other species. The sequences of GIF domain were extracted using the coordinates of the GIF domain and merged into a new sequence matrix. Then, the merged protein sequences were aligned by ClustalW. After aligning the amino acid sequences, gap trimming was performed using the Multiple Alignment Trimming tools of TBtools software with a Site Coverage Cut off parameter set at 0.95. Subsequently, a phylogenetic tree was constructed using MEGA v7 software employing the neighbor-joining (NJ) method with 1000 bootstrap replicates. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches. The Dayhoff matrix-based method was used to calculate evolutionary distances, which were expressed as the number of amino acid substitutions per site. Ambiguous positions were excluded for each pair of sequences using the pairwise deletion option. TBtools and iTOL online website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used to visualize the phylogenetic tree.\u003c/p\u003e \u003cp\u003eAdditionally, we used the MEME tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite.org/\u003c/span\u003e\u003cspan address=\"http://meme-suite.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to classify and analyze the conserved motifs of each GIF protein sequence. We set the maximum motif number was 5 and other parameters are default settings..\u003c/p\u003e \u003cp\u003e \u003cb\u003eCollinearity analysis of multi-species\u003c/b\u003e \u003cb\u003eGIFs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe BLASTP alignment was used to identify orthologous pairs between \u003cem\u003eP. euphratica\u003c/em\u003e and six other species (\u003cem\u003eP. pruinose, P. trichocarpa, P. deltoides, S. sinopurpurea, S. suchowensis\u003c/em\u003e, and \u003cem\u003eA. thaliana\u003c/em\u003e). Then, the collinear blocks between \u003cem\u003eP. euphratica\u003c/em\u003e and each other species of \u003cem\u003eP. deltoide\u003c/em\u003e, \u003cem\u003eP. trichocarpa\u003c/em\u003e, \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eS. sinopurpurea\u003c/em\u003e and \u003cem\u003eS. sinopurpurea\u003c/em\u003e were identified using MCscan software and visualized using JCVI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://zenodo.org/record/31631/\u003c/span\u003e\u003cspan address=\"https://zenodo.org/record/31631/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePromoter analysis of\u003c/b\u003e \u003cb\u003eGIF\u003c/b\u003e \u003cb\u003epromoters\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe upstream 2000 base pair (bp) sequences apart from the transcription start sites of these \u003cem\u003ePeGIFs\u003c/em\u003e genes were identified as potential promoters using TBtools. Subsequently, the \u003cem\u003ecis\u003c/em\u003e-elements within each promoter were identified using PlantCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq for heteromorphic leaves\u003c/h2\u003e \u003cp\u003eA total of 12 samples for four leaf shapes in cultivated forests, including linear (Li), lanceolate (La), ovate (Ov) and broad-ovate (Bo) leaves, were collected across the development of leaf age. These samples were collected at various stages of leaf development. Leaf age was categorized into three periods based on field sampling and observation. The first period (P1) was defined as the first day when the leaf blades started unfolding. This was followed by a transitional period (P2) occurring on the 15th day when there was an increase in leaf area. Finally, the third period (P3) occurred on the 30th day when leaves reached maturity. Each type of heteromorphic leaves with different leaf ages was replicated three times for sampling. The napkin was used to delicately clean the leaves, which were then rapidly frozen in liquid nitrogen and stored at an ultra-low temperature of -80℃ in a refrigerator for RNA-seq analysis (the dataset has been made available to the public for access[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and preservation through the National Genomics Data Center (NGDC, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngdc.cncb.ac.cn/\u003c/span\u003e\u003cspan address=\"https://ngdc.cncb.ac.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), under project number PRJCA005959).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of transcriptomes using short reads from Illumina sequencing.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs part of the study, we conducted whole transcriptome sequencing using mRNA-Seq on an Illumina Hiseq X-Ten platform, following the protocol recommended by the vendor. To assess the relative abundance of the annotated genes from \u003cem\u003eP. euphratica\u003c/em\u003e, we employed HISAT2 (version 2.0.4) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] to align the clean reads against our reference genome. The gene expression was quantified with FPKM using StringTie [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eValidation of\u003c/b\u003e \u003cb\u003ePeGIFs\u003c/b\u003e \u003cb\u003eusing quantitative reverse-transcription polymerase chain reaction (qRT-PCR)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe heteromorphic foliage was collected from different canopies and stored in an ultra-low temperature refrigerator at -80\u0026deg;C after being rapidly frozen with liquid nitrogen. The procedure followed the methodology described in a previous publication [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. \u003cem\u003eActin\u003c/em\u003e gene was used as the endogenous control. Each reaction was performed in biological triplicates, and CT values obtained through qRT-PCR were analyzed using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method to calculate relative fold change values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePlant growth conditions, treatments, and sampling\u003c/h2\u003e \u003cp\u003eAll \u003cem\u003eArabidopsis thaliana\u003c/em\u003e mutants and transgenic plants that were used in this study were from the Columbia (Col-0) ecotype. The Arabidopsis seeds were sown on moist soil, stratified at 4 ℃ for 3 days, and then transferred to a growth room with a temperature of 21 ℃ and a photoperiod of 16 hours light/8 hours darkness. \u003cem\u003eAtgif1\u003c/em\u003e (SALK_208834C) seeds were obtained from the AraShare. The leaves of \u003cem\u003eP. euphratica\u003c/em\u003e were collected from the forest located at the eastern entrance of Tarim University.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCloning, construction of transgenic plants\u003c/h3\u003e\n\u003cp\u003eThe laboratory have preserved \u003cem\u003eEscherichia coli\u003c/em\u003e (DH5α)、\u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e (GV3101)、overexpressed vector (pGreenII 0179). The RNA was extracted from \u003cem\u003eP. euphratica\u003c/em\u003e Bo leaves using Trizol (Invitrogen), followed by cDNA synthesis using the M5 Sprint qPCR RT kit with gDNA remover (Mei5 Biotechnology). The full-length coding regions of \u003cem\u003ePeGIF3\u003c/em\u003e genes lacking a stop codon were amplified from cDNA, or plasmid using Phanta Max Super-Fidelity DNA polymerase (Vazyme) to ensure high fidelity. Subsequently introduced into a yellow fluorescent protein (YFP) vector to generate a construct using the T4 DNA Ligase (Sangon Biotech). The \u003cem\u003eGIF\u003c/em\u003e::YFP fusion was inserted into the pGreenII 0179 vector, which contained a CaMV 35S promoter and a NOS terminator cassette. The floral dip method was utilized for the transformation of \u003cem\u003eArabidopsis\u003c/em\u003e plants [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The overexpression of \u003cem\u003ePeGIF3\u003c/em\u003e was established with a wild-type background. The single-insertion homozygous T3 lines of the \u003cem\u003ePeGIF3\u003c/em\u003e complement were carefully chosen and established in the \u003cem\u003eatgif1\u003c/em\u003e mutant background.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSubcellular localization of\u003c/b\u003e \u003cb\u003ePeGIF3\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTransform the constructed 35S:\u003cem\u003ePeGIF3\u003c/em\u003e-YFP vector into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101. Reconstitute the strain harboring the target plasmid(NLS-mcherry) in LB medium supplemented with appropriate antibiotics for overnight cultivation. Inoculate the bacterial solution obtained in the second step into fresh LB medium, simultaneously adding acetosyringone, and agitate until the bacteria reach an optical density (OD600) of 1.0-1.2. The supernatant should be discarded by centrifugation, and the bacteria should be resuspended in infection fluid (0.01M MES (pH\u0026thinsp;=\u0026thinsp;5.6), 0.01M MgCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO and 50 \u0026micro;M acetosyringone) until the OD value reaches approximately 1.0. Allow it to remain undisturbed for a duration of 3 hours in a lightless environment. The target bacterial was combined with the NLS-mcherry in equal proportions, and tobacco were inoculated using a syringe. The treated plants were kept in darkness for 12 hours and subsequently incubated under normal conditions for 36 hours. The underlying epidermis of tobacco (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e) was revealed in a dark environment and examined using a laser scanning confocal microscope (Nikon eclipse Ti2). The microscope was excited by a 488 nm laser and emitted signals were detected within the range of 500\u0026ndash;550 nm.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of leaf cell number\u003c/h2\u003e \u003cp\u003eThe leaf cross-section chosen for anatomical analysis was carefully selected to encompass the widest point of the primary vein and subsequently fixed using FAA solution. The paraffin section method was employed to convert these into permanent film [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The samples were subsequently examined and imaged using a scanning electron microscope (OPLENIC CORP). Cells present in the pericycle to the leaf margin were enumerated. The statistical analyses were conducted using Graphpad Prism 9 software. The least significant difference test was employed to determine statistically significant differences between means at a significance level of \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 32160355).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026acute;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYY, JS and CQ, carried out the experiment, collected and organized data and wrote the manuscript. PJ and ZW participated in designing the experiment and directed the study. ZL and ZW, reviewed the manuscript. YY and ZW, helped organize data. PJ, helped do the experiment. ZL and ZW, corresponding author, raised the hypothesis underlying this work, designed the experiment, and helped organize the manuscript structure. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Natural Science Foundation of China, grant number 32371838; the Biological Safety and Genetic Resources Management Project of the Science and Technology Development Center of the National Forestry and Grassland Administration, grant number KJZXSA202303 and Xinjiang Production and Construction Corps Regional Innovation Guidance Program project, grant number 2021BB010.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-seq data for \u003cem\u003eP. euphratica\u003c/em\u003e\u0026apos;s heteromorphic leaves used in this study have been submitted to the NGDC (National Genomics Data Center, https://ngdc.cncb.ac.cn/) under the BioProject accession number PRJCA005959.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBradshaw H, Ceulemans R, Davis J, Stettler R: \u003cstrong\u003eEmerging model systems in plant biology: poplar (Populus) as a model forest tree\u003c/strong\u003e. \u003cem\u003eJournal of Plant Growth Regulation \u003c/em\u003e2000, \u003cstrong\u003e19\u003c/strong\u003e(3):306-313.\u003c/li\u003e\n\u003cli\u003eBrunner AM, Busov VB, Strauss SH: \u003cstrong\u003ePoplar genome sequence: functional genomics in an ecologically dominant plant species\u003c/strong\u003e. \u003cem\u003eTrends in plant science \u003c/em\u003e2004, \u003cstrong\u003e9\u003c/strong\u003e(1):49-56.\u003c/li\u003e\n\u003cli\u003eTaylor G: \u003cstrong\u003ePopulus:\u003cem\u003e Arabidopsis\u003c/em\u003e for forestry. Do we need a model tree?\u003c/strong\u003e \u003cem\u003eAnnals of Botany \u003c/em\u003e2002, \u003cstrong\u003e90\u003c/strong\u003e(6):681-689.\u003c/li\u003e\n\u003cli\u003eTuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S, Rombauts S, Salamov A: \u003cstrong\u003eThe genome of black cottonwood, \u003cem\u003ePopulus trichocarpa\u003c/em\u003e (Torr. \u0026amp; Gray)\u003c/strong\u003e. \u003cem\u003eScience \u003c/em\u003e2006, \u003cstrong\u003e313\u003c/strong\u003e(5793):1596-1604.\u003c/li\u003e\n\u003cli\u003eQiu Q, Ma T, Hu Q, Liu B, Wu Y, Zhou H, Wang Q, Wang J, Liu J: \u003cstrong\u003eGenome-scale transcriptome analysis of the desert poplar, \u003cem\u003ePopulus euphratica\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003eTree physiology \u003c/em\u003e2011, \u003cstrong\u003e31\u003c/strong\u003e(4):452-461.\u003c/li\u003e\n\u003cli\u003eSong Z, Ni X, Yao J, Wang F: \u003cstrong\u003eProgress in studying heteromorphic leaves in \u003cem\u003ePopulus euphratica\u003c/em\u003e: leaf morphology, anatomical structure, development regulation and their ecological adaptation to arid environments\u003c/strong\u003e. \u003cem\u003ePlant signaling \u0026amp; behavior \u003c/em\u003e2021, \u003cstrong\u003e16\u003c/strong\u003e(4):1870842.\u003c/li\u003e\n\u003cli\u003eLiu Y, Li X, Chen G, Li M, Liu M, Liu D: \u003cstrong\u003eEpidermal micromorphology and mesophyll structure of \u003cem\u003ePopulus euphratica\u003c/em\u003e heteromorphic leaves at different development stages\u003c/strong\u003e. \u003cem\u003ePLoS One \u003c/em\u003e2015, \u003cstrong\u003e10\u003c/strong\u003e(9):e0137701.\u003c/li\u003e\n\u003cli\u003eHao J, Yue N, Zheng C: \u003cstrong\u003eAnalysis of changes in anatomical characteristics and physiologic features of heteromorphic leaves in a desert tree, \u003cem\u003ePopulus euphratica\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003eActa Physiologiae Plantarum \u003c/em\u003e2017, \u003cstrong\u003e39\u003c/strong\u003e:1-11.\u003c/li\u003e\n\u003cli\u003eKim JH, Tsukaya H: \u003cstrong\u003eRegulation of plant growth and development by the \u003cem\u003eGROWTH-REGULATING FACTOR\u003c/em\u003e and \u003cem\u003eGRF-INTERACTING FACTOR\u003c/em\u003e duo\u003c/strong\u003e. \u003cem\u003eJournal of experimental botany \u003c/em\u003e2015, \u003cstrong\u003e66\u003c/strong\u003e(20):6093-6107.\u003c/li\u003e\n\u003cli\u003eKim JH, Kende H: \u003cstrong\u003eA transcriptional coactivator, \u003cem\u003eAtGIF1\u003c/em\u003e, is involved in regulating leaf growth and morphology in \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003eProc Natl Acad Sci U S A \u003c/em\u003e2004, \u003cstrong\u003e101\u003c/strong\u003e(36):13374-13379.\u003c/li\u003e\n\u003cli\u003eHoriguchi G, Kim GT, Tsukaya H: \u003cstrong\u003eThe transcription factor \u003cem\u003eAtGRF5\u003c/em\u003e and the transcription coactivator\u003cem\u003e AN3\u003c/em\u003e regulate cell proliferation in leaf primordia of\u003cem\u003e Arabidopsis thaliana\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003eThe Plant journal : for cell and molecular biology \u003c/em\u003e2005, \u003cstrong\u003e43\u003c/strong\u003e(1):68-78.\u003c/li\u003e\n\u003cli\u003eVercruyssen L, Verkest A, Gonzalez N, Heyndrickx KS, Eeckhout D, Han SK, J\u0026eacute;gu T, Archacki R, Van Leene J, Andriankaja M\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003e\u003cem\u003eANGUSTIFOLIA3\u003c/em\u003e binds to SWI/SNF chromatin remodeling complexes to regulate transcription during \u003cem\u003eArabidopsis\u003c/em\u003e leaf development\u003c/strong\u003e. \u003cem\u003eThe Plant cell \u003c/em\u003e2014, \u003cstrong\u003e26\u003c/strong\u003e(1):210-229.\u003c/li\u003e\n\u003cli\u003eErcoli MF, Ferela A, Debernardi JM, Perrone AP, Rodriguez RE, Palatnik JF: \u003cstrong\u003e\u003cem\u003eGIF\u003c/em\u003e Transcriptional Coregulators Control Root Meristem Homeostasis\u003c/strong\u003e. \u003cem\u003eThe Plant cell \u003c/em\u003e2018, \u003cstrong\u003e30\u003c/strong\u003e(2):347-359.\u003c/li\u003e\n\u003cli\u003eLi J, Pan W, Zhang S, Ma G, Li A, Zhang H, Liu L: \u003cstrong\u003eA rapid and highly efficient sorghum transformation strategy using GRF4\u003c/strong\u003e\u003cstrong\u003e‐\u003c/strong\u003e\u003cstrong\u003eGIF1/ternary vector system\u003c/strong\u003e. \u003cem\u003eThe Plant Journal \u003c/em\u003e2023.\u003c/li\u003e\n\u003cli\u003eLee BH, Ko JH, Lee S, Lee Y, Pak JH, Kim JH: \u003cstrong\u003eThe Arabidopsis\u003cem\u003e GRF-INTERACTING FACTOR\u003c/em\u003e gene family performs an overlapping function in determining organ size as well as multiple developmental properties\u003c/strong\u003e. \u003cem\u003ePlant physiology \u003c/em\u003e2009, \u003cstrong\u003e151\u003c/strong\u003e(2):655-668.\u003c/li\u003e\n\u003cli\u003eLiu Y, Guo P, Wang J, Xu ZY: \u003cstrong\u003eGrowth\u003c/strong\u003e\u003cstrong\u003e‐\u003c/strong\u003e\u003cstrong\u003eregulating factors: conserved and divergent roles in plant growth and development and potential value for crop improvement\u003c/strong\u003e. \u003cem\u003eThe Plant Journal \u003c/em\u003e2023, \u003cstrong\u003e113\u003c/strong\u003e(6):1122-1145.\u003c/li\u003e\n\u003cli\u003eLiang G, He H, Li Y, Wang F, Yu D: \u003cstrong\u003eMolecular mechanism of microRNA396 mediating pistil development in \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003ePlant physiology \u003c/em\u003e2014, \u003cstrong\u003e164\u003c/strong\u003e(1):249-258.\u003c/li\u003e\n\u003cli\u003eDuan P, Ni S, Wang J, Zhang B, Xu R, Wang Y, Chen H, Zhu X, Li Y: \u003cstrong\u003eRegulation of \u003cem\u003eOsGRF4\u003c/em\u003e by\u003cem\u003e OsmiR396\u003c/em\u003e controls grain size and yield in rice\u003c/strong\u003e. \u003cem\u003eNature plants \u003c/em\u003e2015, \u003cstrong\u003e2\u003c/strong\u003e:15203.\u003c/li\u003e\n\u003cli\u003eQin L, Chen H, Wu Q, Wang X: \u003cstrong\u003eIdentification and exploration of the GRF and GIF families in maize and foxtail millet\u003c/strong\u003e. \u003cem\u003ePhysiology and Molecular Biology of Plants \u003c/em\u003e2022, \u003cstrong\u003e28\u003c/strong\u003e(9):1717-1735.\u003c/li\u003e\n\u003cli\u003eZhang D, Sun W, Singh R, Zheng Y, Cao Z, Li M, Lunde C, Hake S, Zhang Z: \u003cstrong\u003e\u003cem\u003eGRF-interacting factor1\u003c/em\u003e Regulates Shoot Architecture and Meristem Determinacy in Maize\u003c/strong\u003e. \u003cem\u003eThe Plant cell \u003c/em\u003e2018, \u003cstrong\u003e30\u003c/strong\u003e(2):360-374.\u003c/li\u003e\n\u003cli\u003eAi G, Zhang D, Huang R, Zhang S, Li W, Ahiakpa JK, Zhang J: \u003cstrong\u003eGenome-Wide Identification and Molecular Characterization of the \u003cem\u003eGrowth-Regulating Factors-Interacting Factor\u003c/em\u003e Gene Family in Tomato\u003c/strong\u003e. \u003cem\u003eGenes \u003c/em\u003e2020, \u003cstrong\u003e11\u003c/strong\u003e(12).\u003c/li\u003e\n\u003cli\u003eWu ZJ, Wang WL, Zhuang J: \u003cstrong\u003eDevelopmental processes and responses to hormonal stimuli in tea plant (\u003cem\u003eCamellia sinensis\u003c/em\u003e) leaves are controlled by \u003cem\u003eGRF\u003c/em\u003e and \u003cem\u003eGIF\u003c/em\u003e gene families\u003c/strong\u003e. \u003cem\u003eFunctional \u0026amp; integrative genomics \u003c/em\u003e2017, \u003cstrong\u003e17\u003c/strong\u003e(5):503-512.\u003c/li\u003e\n\u003cli\u003eZhang ZS, Zeng QY, Liu YJ: \u003cstrong\u003eFrequent ploidy changes in Salicaceae indicates widespread sharing of the salicoid whole genome duplication by the relatives of Populus L. and Salix L\u003c/strong\u003e. \u003cem\u003eBMC plant biology \u003c/em\u003e2021, \u003cstrong\u003e21\u003c/strong\u003e(1):535.\u003c/li\u003e\n\u003cli\u003eLiu L, Xu W, Hu X, Liu H, Lin Y: \u003cstrong\u003eW-box and G-box elements play important roles in early senescence of rice flag leaf\u003c/strong\u003e. \u003cem\u003eScientific reports \u003c/em\u003e2016, \u003cstrong\u003e6\u003c/strong\u003e:20881.\u003c/li\u003e\n\u003cli\u003eDebernardi JM, Mecchia MA, Vercruyssen L, Smaczniak C, Kaufmann K, Inze D, Rodriguez RE, Palatnik JF: \u003cstrong\u003ePost-transcriptional control of\u003cem\u003e GRF \u003c/em\u003etranscription factors by microRNA miR396 and \u003cem\u003eGIF\u003c/em\u003e co-activator affects leaf size and longevity\u003c/strong\u003e. \u003cem\u003eThe Plant journal : for cell and molecular biology \u003c/em\u003e2014, \u003cstrong\u003e79\u003c/strong\u003e(3):413-426.\u003c/li\u003e\n\u003cli\u003eYan S, Zou G, Li S, Wang H, Liu H, Zhai G, Guo P, Song H, Yan C, Tao Y: \u003cstrong\u003eSeed size is determined by the combinations of the genes controlling different seed characteristics in rice\u003c/strong\u003e. \u003cem\u003eTAG Theoretical and applied genetics Theoretische und angewandte Genetik \u003c/em\u003e2011, \u003cstrong\u003e123\u003c/strong\u003e(7):1173-1181.\u003c/li\u003e\n\u003cli\u003eLi S, Gao F, Xie K, Zeng X, Cao Y, Zeng J, He Z, Ren Y, Li W, Deng Q\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eThe \u003cem\u003eOsmiR396c-OsGRF4-OsGIF1\u003c/em\u003e regulatory module determines grain size and yield in rice\u003c/strong\u003e. \u003cem\u003ePlant biotechnology journal \u003c/em\u003e2016, \u003cstrong\u003e14\u003c/strong\u003e(11):2134-2146.\u003c/li\u003e\n\u003cli\u003eXie Y, Skytting B, Nilsson G, Grimer RJ, Mangham CD, Fisher C, Shipley J, Bjerkehagen B, Myklebost O, Larsson O: \u003cstrong\u003eThe SYT-SSX1 fusion type of synovial sarcoma is associated with increased expression of cyclin A and D1. A link between t(X;18)(p11.2; q11.2) and the cell cycle machinery\u003c/strong\u003e. \u003cem\u003eOncogene \u003c/em\u003e2002, \u003cstrong\u003e21\u003c/strong\u003e(37):5791-5796.\u003c/li\u003e\n\u003cli\u003eNelissen H, Eeckhout D, Demuynck K, Persiau G, Walton A, van Bel M, Vervoort M, Candaele J, De Block J, Aesaert S\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eDynamic Changes in \u003cem\u003eANGUSTIFOLIA3\u003c/em\u003e Complex Composition Reveal a Growth Regulatory Mechanism in the Maize Leaf\u003c/strong\u003e. \u003cem\u003eThe Plant cell \u003c/em\u003e2015, \u003cstrong\u003e27\u003c/strong\u003e(6):1605-1619.\u003c/li\u003e\n\u003cli\u003eZhang Z, Chen Y, Zhang J, Ma X, Li Y, Li M, Wang D, Kang M, Wu H, Yang Y\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eImproved genome assembly provides new insights into genome evolution in a desert poplar (\u003cem\u003ePopulus euphratica\u003c/em\u003e)\u003c/strong\u003e. \u003cem\u003eMolecular ecology resources \u003c/em\u003e2020, \u003cstrong\u003e20\u003c/strong\u003e(3).\u003c/li\u003e\n\u003cli\u003eXue L, Wu H, Chen Y, Li X, Hou J, Lu J, Wei S, Dai X, Olson MS, Liu J: \u003cstrong\u003eEvidences for a role of two Y-specific genes in sex determination in \u003cem\u003ePopulus deltoides\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003eNature Communications \u003c/em\u003e2020, \u003cstrong\u003e11\u003c/strong\u003e(1):5893.\u003c/li\u003e\n\u003cli\u003eZhou R, Macaya-Sanz D, Carlson CH, Schmutz J, Jenkins JW, Kudrna D, Sharma A, Sandor L, Shu S, Barry K\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eA willow sex chromosome reveals convergent evolution of complex palindromic repeats\u003c/strong\u003e. \u003cem\u003eGenome biology \u003c/em\u003e2020, \u003cstrong\u003e21\u003c/strong\u003e(1):38.\u003c/li\u003e\n\u003cli\u003eDai X, Hu Q, Cai Q, Feng K, Ye N, Tuskan GA, Milne R, Chen Y, Wan Z, Wang Z\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eThe willow genome and divergent evolution from poplar after the common genome duplication\u003c/strong\u003e. \u003cem\u003eCell research \u003c/em\u003e2014, \u003cstrong\u003e24\u003c/strong\u003e(10):1274-1277.\u003c/li\u003e\n\u003cli\u003eWu Z, Jiang Z, Li Z, Jiao P, Zhai J, Liu S, Han X, Zhang S, Sun J, Gai Z: \u003cstrong\u003eMulti-omics analysis reveals spatiotemporal regulation and function of heteromorphic leaves in Populus\u003c/strong\u003e. \u003cem\u003ePlant physiology \u003c/em\u003e2023, \u003cstrong\u003e192\u003c/strong\u003e(1):188-204.\u003c/li\u003e\n\u003cli\u003ePertea M, Kim D, Pertea GM, Leek JT, Salzberg SL: \u003cstrong\u003eTranscript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown\u003c/strong\u003e. \u003cem\u003eNature protocols \u003c/em\u003e2016, \u003cstrong\u003e11\u003c/strong\u003e(9):1650-1667.\u003c/li\u003e\n\u003cli\u003eKovaka S, Zimin AV, Pertea GM, Razaghi R, Salzberg SL, Pertea M: \u003cstrong\u003eTranscriptome assembly from long-read RNA-seq alignments with StringTie2\u003c/strong\u003e. \u003cem\u003eGenome biology \u003c/em\u003e2019, \u003cstrong\u003e20\u003c/strong\u003e(1):1-13.\u003c/li\u003e\n\u003cli\u003eLiu C, Hao J, Qiu M, Pan J, He Y: \u003cstrong\u003eGenome-wide identification and expression analysis of the MYB transcription factor in Japanese plum (\u003cem\u003ePrunus salicina\u003c/em\u003e)\u003c/strong\u003e. \u003cem\u003eGenomics \u003c/em\u003e2020, \u003cstrong\u003e112\u003c/strong\u003e(6):4875-4886.\u003c/li\u003e\n\u003cli\u003eZhang X, Henriques R, Lin SS, Niu QW, Chua NH: \u003cstrong\u003eAgrobacterium-mediated transformation of \u003cem\u003eArabidopsis\u003c/em\u003e thaliana using the floral dip method\u003c/strong\u003e. \u003cem\u003eNature protocols \u003c/em\u003e2006, \u003cstrong\u003e1\u003c/strong\u003e(2):641-646.\u003c/li\u003e\n\u003cli\u003eFolkers U, Kirik V, Sch\u0026ouml;binger U, Falk S, Krishnakumar S, Pollock MA, Oppenheimer DG, Day I, Reddy AS, J\u0026uuml;rgens G\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eThe cell morphogenesis gene \u003cem\u003eANGUSTIFOLIA\u003c/em\u003e encodes a CtBP/BARS-like protein and is involved in the control of the microtubule cytoskeleton\u003c/strong\u003e. \u003cem\u003eThe EMBO journal \u003c/em\u003e2002, \u003cstrong\u003e21\u003c/strong\u003e(6):1280-1288.\u003c/li\u003e\n\u003cli\u003eTsukaya H, Naito S, R\u0026eacute;dei GP, Komeda Y: \u003cstrong\u003eA new class of mutations in \u003cem\u003eArabidopsis thaliana,\u003c/em\u003e acaulis1, affecting the development of both inflorescences and leaves\u003c/strong\u003e. \u003cem\u003eDevelopment (Cambridge, England) \u003c/em\u003e1993, \u003cstrong\u003e118\u003c/strong\u003e(3):751-764.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Salicaceae, Populus euphratica, GIF genes, heteromorphic leaves, regulatory function","lastPublishedDoi":"10.21203/rs.3.rs-3881684/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3881684/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eGIF\u003c/em\u003e(\u003cem\u003eGrowth-Regulating Factors-Interacting Factors\u003c/em\u003e) gene family plays a vital role in regulating plant growth and development, particularly in controlling leaf, seed, and root meristem homeostasis. As an important adaptative trait of heteromorphic leaves in response to desert environment, however, the regulatory mechanism of heteromorphic leaves by \u003cem\u003eGIF\u003c/em\u003e genes in \u003cem\u003ePopulus euphratica\u003c/em\u003e remains unknown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur study aimed to identify and characterize the \u003cem\u003eGIF\u003c/em\u003e genes in \u003cem\u003ePopulus euphratica\u003c/em\u003e and other five Salicaceae species to investigate their role in regulating heteromorphic leaf development. We identified and characterized a total of 27\u003cem\u003e GIF\u003c/em\u003e genes across six Salicaceae species (\u003cem\u003eP. euphratica\u003c/em\u003e, \u003cem\u003ePopulus pruinose\u003c/em\u003e, \u003cem\u003ePopulus deltoides\u003c/em\u003e, \u003cem\u003ePopulus trichocarpa\u003c/em\u003e, \u003cem\u003eSalix sinopurpurea\u003c/em\u003e, and \u003cem\u003eSalix suchowensis\u003c/em\u003e) at the genome-wide level. Then, the comparative genomic analysis among these species suggested that the expansion\u003cem\u003e \u003c/em\u003eof \u003cem\u003eGIFs\u003c/em\u003emay be derived the specific Salicaceae whole-genome duplication event after their divergence from Arabidopsis. Elements analysis suggested that \u003cem\u003eGIFs\u003c/em\u003ewere suffering from diverse regulation by hormones and environment clues. Furthermore, the expression data of \u003cem\u003ePeGIFs\u003c/em\u003e in heteromorphic leaves, combined with functional information on \u003cem\u003eGIF\u003c/em\u003e genes in \u003cem\u003eArabidopsis thaliana,\u003c/em\u003e indicate the role of \u003cem\u003ePeGIFs\u003c/em\u003e in regulating leaf development of \u003cem\u003eP. euphratica\u003c/em\u003e, especially \u003cem\u003ePeGIFs\u003c/em\u003e contain several auxin-related cis-acting elements such as TGA-box. By heterologous expression the \u003cem\u003ePeGIF3\u003c/em\u003e gene in both wild-type plants (Col-0) and \u003cem\u003egif1\u003c/em\u003emutant of \u003cem\u003eA. thaliana\u003c/em\u003e, a significant difference in leaf expansion along the medial-lateral axis, as well as an increased number of leaf cells, along with the increased number of leaf cells was observed between the overexpressed plants and the wild type.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results indicated that \u003cem\u003ePeGIF3\u003c/em\u003e enhances leaf cell proliferation by modulating transcriptional processes, thereby resulting in the expansion of the central-lateral region of the leaf. Our findings not only provide global insights into the evolutionary features of Salicaceae GIFs, but also reveal the regulatory mechanism of \u003cem\u003ePeGIF3\u003c/em\u003e in heteromorphic leaves in \u003cem\u003eP. euphratica\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Comparative genomic analysis of the Growth Regulating Factors-Interacting Factors (GIFs) in six Salicaceae species and functional analysis of PeGIF3 reveals their regulatory role in Populus heteromorphic leaves","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-23 12:03:14","doi":"10.21203/rs.3.rs-3881684/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-02-12T13:54:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-11T11:53:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33919876-7c07-498f-bc24-63ef5d460d86","date":"2024-02-06T07:35:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-04T02:04:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b978de84-cb9f-4a0e-afbb-b76556e40503","date":"2024-01-28T01:52:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5050d67b-20da-48c9-a755-6ed044c42294","date":"2024-01-27T14:26:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-27T14:21:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-27T14:19:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-01-21T11:37:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-21T11:34:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2024-01-20T13:44:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3943275f-58f1-46af-a816-84a00a726389","owner":[],"postedDate":"January 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-03-29T13:50:06+00:00","versionOfRecord":{"articleIdentity":"rs-3881684","link":"https://doi.org/10.1186/s12864-024-10221-5","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2024-03-28 00:00:00","publishedOnDateReadable":"March 28th, 2024"},"versionCreatedAt":"2024-01-23 12:03:14","video":"","vorDoi":"10.1186/s12864-024-10221-5","vorDoiUrl":"https://doi.org/10.1186/s12864-024-10221-5","workflowStages":[]},"version":"v1","identity":"rs-3881684","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3881684","identity":"rs-3881684","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}