Genome-wide investigation of the AP2/ERF superfamily and their expression under salt stress in Chinese willow (Salix matsudana)

Genome-wide investigation of the AP2/ERF superfamily and their expression under salt stress in Chinese willow (Salix matsudana) | 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 Genome-wide investigation of the AP2/ERF superfamily and their expression under salt stress in Chinese willow ( Salix matsudana ) Yanhong Chen, Yuna Jiang, Guoyuan Liu, Chunmei Yu, Bolin Lian, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.24667/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Apr, 2021 Read the published version in PeerJ → Version 1 posted You are reading this latest preprint version Abstract Background: AP2/ERF transcription factors (TFs) play indispensable roles in plant growth, development, and especially in various abiotic stresses responses. The AP2/ERF TF family has been discovered and classified in more than 50 species, including model plants, horticulture plants, crops, and trees. Many AP2/ERF TFs from various species have been functionally characterized. However, little is known about the AP2/ERF gene family of Chinese willow ( Salix matsudana ), which is a tetraploid ornamental tree species that is widely planted and is also considered a species that can improve the soil salinity of coastal beaches. The recently produced whole genome sequencing data of Salix matsudana allowed us to conduct an evolutionary analysis on the AP2/ERF genes during polyploidization and study the genome-wide expression profiles of AP2/ERF genes in Salix matsudana under salt stress. Results: In this study, 364 AP2/ERF genes of Salix matsudana (SmAP2/ERF) were identified and renamed according to the chromosomal location of the SmAP2/ERF genes. After phylogenetic analysis with known categories of genes from other species, the AP2/ERF genes were divided into three subfamilies: AP2 (55 members), ERF (301 members), and RAV (six members). Two Soloist genes were also identified. Gene structure and conserved motifs were analyzed in AP2/ERF genes, and introns were not found in most genes of the ERF subfamily, although some unique motifs were found to be important for the function of AP2/ERF genes. Syntenic relationships between the SmAP2/ERF genes and AP2/ERF genes from other species were also investigated to elucidate their evolutionary relationships during polyploidization. Moreover, analyses on the expression profiles under salt stress were also conducted. Conclusion: The progenitors of Salix matsudana underwent whole genome duplication not more than 10 Mya. Synteny analysis with other species showed macrosynteny between willow and poplar AP2/ERF genes and that Salix matsudana is genetically more closely related to Populus trichocarpa than to Salix purpurea . Further investigation of the AP2/ERF TFs demonstrated that differential expression patterns during salt stress and this information can help reveal the mechanism of salt tolerance regulation in Salix matsudana . Epigenetics & Genomics Salix matsudana SmAP2/ERF Genome-wide Salt tolerance Expression patterns Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background AP ETALA 2/ e thylene- r esponsive element binding f actors (AP2/ERF) are important transcription factors (TFs) coded by genes from the AP2/ERF superfamily. All of the members of this superfamily possess AP2 domains and, according to the number and structure of AP2 domains, the superfamily is divided into several categories, including AP2, ERF, RAV, and Soloist [ 1 ]. Most of the AP2 gene family members have two AP2 domains and can be further divided into AP2 and ANT groups; ERF family members have only one AP2 domain and can also be subdivided into ERF and DREB subfamilies based on binding motifs in the promoter of downstream genes. Members of the ERF and DREB subfamilies are classified into 12 groups (groups A1–B6). DREB includes groups A1–A6, whereas ERF includes groups B1–B6 [ 1 ]. In addition to one AP2 domain, RAVs also have one B3 domain. The Soloist group contains a single AP2 domain with sequence divergence from the AP2 and ERF families and has less than three members in most species [ 2 ]. The AP2/ERF superfamily is plant-specific and has more than 100 members in many plant species; for example, there are 147 members in Arabidopsis , 200 members in Populus trichocarpa , and more than 500 members in the tetraploid crop Brassica napus [ 1 – 3 ]. Different members play various regulatory roles in plant growth and development, defense response, fruit ripening, and metabolism [ 4 ]. Several recent reports demonstrated functions of AP2/ERF2 TFs in plant development. For example, loss of DRNL function affects gynoecium development [ 5 ]; the function of Populus ERF139 (Potri.013G101100) in xylem cell expansion was characterized by transgenic overexpression and dominant repressor lines of ERF139 [ 6 ]; RhERF1 and RhERF4 play roles in petal abscission in rose [ 7 ]; and a maize AP2/ERF TF, ZmRAP2.7, is involved in brace root development. AP2/ERF TFs such as ZmEREB94 and CitAP2.10 also play important roles in plant metabolism; ZmEREB94 acts as a key regulator of starch synthesis in maize [ 8 ], and CitAP2.10 was characterized as a regulator of (+)-valencene synthesis in sweet orange fruit [ 9 ]. The AP2/ERF superfamily plays major and crucial roles in abiotic stress tolerance, which is why this superfamily has received special attention by plant scientists [ 4 , 10 ]. Through extensive investigation on their regulatory mechanism, people want to elucidate their potential applications in crop improvement [ 10 ]. Members of this superfamily (primarily ERFs and DREBs) have been prominently used to improve stress tolerance in plants. To improve salinity stress tolerance, many genes from different species were identified. IbRAP2-12, an AP2/ERF gene cloned from the salt-tolerant sweet potato, and LkERF-B2 from Larix kaempferi promote tolerance to salt and drought stresses in overexpressing Arabidopsis lines [ 11 , 12 ]. Overexpression of HARDY, an AP2/ERF gene from Arabidopsis , improves drought and salt tolerance by reducing transpiration and sodium uptake in transgenic Trifolium alexandrinum L [ 13 ]. A soybean DREB ortholog, GmDREB1, enhances the salt tolerance in transgenic alfalfa [ 14 ]. Comparative genomic analysis of model plants such as Arabidopsis have provided unprecedented advantages for gene discovery and functional annotation of newly sequenced plant genomes [ 15 – 17 ]. By exploring the available genomic data, AP2/ERF gene families from 50 species were discovered and classified, and provide critical guidance for functional analysis [ 10 ]. For example, in radish, cauliflower, and celery, whole genome identification and classification of AP2/ERF gene family members were carried out; additionally, expression patterns of different members under different stresses were revealed, and the function of candidate genes was verified [ 18 – 20 ]. Salix matsudana Koidz ., a member of Salicaceae, is an important ornamental tree species native to northeastern China [ 21 , 22 ]; it is widely cultivated and considered an important economic plant because of its easy vegetative propagation, rapid growth, and substantial biomass yields. Salix matsudana also plays an important ecological role when grown along Chinese coastal beaches, where the salinity content is high [ 21 ]. This species can improve the beach soil and alleviate salinization. Newly reclaimed beach soil has higher salinity and requires new germplasm with higher salinity tolerance [ 21 ]. Because the AP2/ERF gene family members have regulatory roles in salinity tolerance, whole genome characterization of the AP2/ERF gene family in Salix matsudana will reveal mechanisms underlying stress signal transmission and provide guidance for selection or creation of new germplasm with higher salinity tolerance. In total, 200 and 173 AP2/ERF superfamily genes were identified from two species, Salix arbutifolia and Populus trichocarpa , respectively [ 3 , 23 ]. The Salix matsudana genome was recently sequenced and assembled (unpublished); as a tetraploid, identification of the AP2/ERF gene family will reveal the evolutionary relationship with poplar and other members of Salix , and the molecular mechanisms responsible for salinity stress responses. Results Identification, phylogenetic analysis, and classification of 364 AP2/ERF TF family memebers in Salix matsudana By HMM profile search against the Salix matsudana protein database, a total of 364 full-length AP2/ERF family proteins containing at least one AP2/ERF domain were identified as AP2/ERF superfamily members of Salix matsudana (Fig. 1 ). The name, protein length, molecular weight, and isoelectric point of individual genes are listed in Supplementary Table S1. The phylogenetic relationships of SmAP2/ERF proteins were inferred by multiple sequence alignment of the AP2 domain, which included approximately 50–60 amino acids. The sequence alignment of all AP2/ERF genes showed some conserved amino acids at specific positions, as previously reported (Fig. S1). For example, the WLG element (58th–60th amino acids; 58–60AA) was highly conserved in in the ERF and RAV families; alternatively, in the AP2 family, the conserved sequences from 58–60AA were converted into YLG elements [ 24 ]. In many species, these conserved amino acid profiles contribute to convincing classification of AP2/ERF genes. Basing on multiple sequence alignments of 48 AP2/ERF proteins from Arabidopsis and Populus trichocarpa with known categories and 364 Salix matsudana AP2/ERF proteins, we constructed a phylogenetic tree using the neighbor-joining method to explore the phylogenetic relationships of Salix matsudana AP2/ERF proteins. The phylogenetic tree showed that there were 55 AP2/ERF genes that belong to the AP2 family, with 47 genes that encode proteins with two AP2 domains and eight genes (SmAP2-20, SmAP2-25, SmAP2-29, SmAP2-35, SmAP2-36, SmAP2-40, SmAP2-41 and SmAP2-55) that encode proteins with a single AP2 domain (Fig. 1 ). Additionally, 301 genes that were predicted to encode proteins with a single AP2 domain were members of the ERF family. The ERF family could be further classified into two subfamilies, ERF and DREB. Of the 301 members, 166 and 135 genes belonged to the ERF and DREB subfamilies, respectively. The ERF family genes from Salix matsudana were distributed in B1–B6 subgroups; the DREB family genes from Salix matsudana were classified into A1–A6 subgroups. The gene number and percentage of each subgroup are listed in Fig. 2 and Table S2. Six putative genes were classified as RAV subgroup genes that encode proteins containing one AP2/ERF domain and one B3 domain (Fig. 1 ). Two genes were designated as Soloist genes, whose AP2/ERF-like domain sequences had lower homology compared with other AP2/ERF genes (Fig. 1 ). The AP2/ERF genes number, classification and percentage of different subgroups from five plant species, including the model plant Arabidopsis , Populus , and two Salix plants, are listed in Table S2. The gene name of AP2/ERF genes from Populus trichocarpa and Salix purpurea are listed in Table S3. As a tetraploid plant, the total number (364) of AP2/ERF genes was much larger in Salix matsudana than in the other four species. The number of AP2/ERF genes in Salix matsudana was 2.5-, 1.8-, 1.9-, and 2.1-fold higher than those in A. thaliana [ 1 ], Populus trichocarpa [ 3 ], Salix purpurea , and Salix arbutifolia [ 23 ], respectively. For DREB and ERF subfamilies, the percentage of all AP2/ERF genes in Salix matsudana was similar to those of A. thaliana , Populus trichocarpa , and Salix purpurea , and the percentages of DREB and ERF subfamilies were 38% and 45%, respectively. In Salix arbutifolia , the percentage of DREB (33%) was lower than that of the other four species, whereas the percentage of ERF (50.8%) was higher. In Salix matsudana , the percentage of the AP2 subgroup was highest among all five species (15%) and the numbers of most of gene sub-classifications were doubled, including the Soloist gene; there were two Soloist genes in the Salix matsudana genome. However, no duplications were observed in the RAV subgroup, and only six RAV genes were found in the Salix matsudana genome. Gene Structure And Conserved Motif Analysis To understand the structural diversity of SmAP2/ERF genes in different clades, the intron and exon structures of SmAP2/ERF genes were revealed by inputting Gff3 files into TBtools (Fig. 3 b). A total of 55 genes of the AP2 subfamily had more exons than ERF and other subfamilies. Apart from three exons in the SmAP2-29 and four exons in the SmAP2-20 , other members of the AP2 subfamily contained more than seven exons. The intron number was less than three in many members of the ERF and RAV subfamilies. In total, 215 gene members did not have introns (Fig. 3 b). The exon/intron structures of genes that were classified in the same clade were similar. Many gene pairs were found in the phylogenetic tree that potentially originated from allotetraploid or autotetraploid evolution of Salix matsudana . Many gene pairs (approximately 70%) maintained the same or similar gene structure during Salix matsudana formation, which indicated that the SmAP2/ERF genes were conserved at the DNA level during polyploidization. TF proteins always contain many conserved motifs to activate gene expression. A total of 10 conserved motifs were detected in 364 SmAP2/ERF proteins using the online MEME software, and a block diagram was constructed to characterize SmAP2/ERF protein structure (Fig. 2 c). Motif-4, Motif-1, Motif-2, Motif-3, Motif-5, Motif-7, and Motif-9 were found in the AP2 domain regions. The Motif-5 region covered the region of Motif-4 and Motif-1, whereas Motif-7 included Motif2 and Motif3. Motif-9 is a specific motif that is only found in the second AP2 domain of the AP2 subgroup. Motif-1, Motif-2, Motif-3, and Motif-4 were detected in 90% percent of the ERF subfamily proteins. Thirty proteins of the ERF subfamily lacked one or two motifs of Motif-1–4. An extreme example is SmERF B2-13 , which only had Motif-2. Motif-6, Motif-8, and Motif-10 are motifs located outside of the AP2 domain. Motif-6 was primarily found in the AP2 subfamily with only one exception, SmERF B4-4 , which was in the ERF-B4 clade. In the AP2 subfamily, members with two AP2 domains had Motif-6 located between the two AP2 domains. Motif-8 was found in 69 proteins of the AP2/ERF family, and its location was adjacent to the carboxyl terminal of Motif-3. Many proteins from the DREB-A1, DREB-A4, DREB-A5 clades had Motif-8. Motif-10 was found in 62 proteins of the AP2/ERF family, with 61 proteins from the ERF subfamily and only one from the AP2 subfamily. Motif-10 was mostly distributed on the proteins from the ERF-B3, DREB-A2, and DREB-A4 clades. The functions of these three motifs need to be elucidated by further experimental analysis. Besides protein SmAP2-20, the entire AP2 domain was distributed in the amino terminal or in the middle position of the proteins. In the two Soloist genes, only one motif, Motif-2, was found. The conserved motif composition and gene structure of the same subfamily were similar, thus verifying the reliability of the phylogenetic tree classification. Chromosome distribution and duplication of SmAP2/ERF superfamily genes The chromosome location of the identified SmAP2/ERF genes was constructed using TBtools. In total, 310 genes from the AP2/ERF superfamily were unevenly distributed on 38 chromosomes (Fig. 4 ); 54 other genes located on scaffolds were not illustrated in Fig. 4 . The chromosome with the largest number of AP2/ERF genes was Chr21, which had 22 genes. Only one AP2/ERF gene each was located on Chr14 and Chr36. On the four chromosomes Chr1, Chr3, Chr22, and Chr27, only two AP2/ERF genes were found. In 38 chromosomes, most of the AP2/ERF genes from different subgroups were arbitrarily distributed, such as five of six RAV genes located on Chr15, Chr37, Chr34, Chr31, and Chr11. Moreover, the two Soloist genes distributed on Chr29 and Chr5. However, SmERF B3 subgroup members clustered together with three genes as a cluster unit. We found 12 clusters in 12 chromosomes (Fig. 4 ), which accounted for 62% of the whole SmERF B3 subgroup. In addition, we also analyzed the tandem duplication events (TDs) of the AP2/ERF genes located within in the 200-kb range of chromosomal regions of the Salix matsudana genome. Eleven TD regions, which included 23 SmAP2/ERF genes, clustered into 11 linkage groups (LGs) of the Salix matsudana genome (Fig. 4 ). LGs that contained cluster repeat genes were Chr7, Chr8, Chr10, Chr13, Chr17, Chr19, Chr21, Chr24, Chr28, Chr31, and Chr33. All genes of the repeat clusters were localized within a genomic segment of less than 20 Kb; for example, SmDREB A4-20 and SmDREB A4-19 were localized on a 3.6-Kb segment of Chr24. On Chr8, three genes clusters (SmERF B3-6, SmERF B3-7, and SmERF B3-8) located on a less than 12-Kb segment. In 11 tandem repeats, six came from the SmERF B3 subgroup, two came from the SmDREB A4 subgroup, and one each came from the SmDREB A1 and AP2 subgroups. SmERF B3-40 and SmERF B3-39 tandem repeat pairs had 97% protein sequence identity. In addition to tandem duplications, many segmental duplication events (SDs) were found in Salix matsudana by MCScanX (Fig. 5 , Table S4). We found a total of 28,348 collinear gene pairs (not shown) in the Salix matsudana genome, from which 298 AP2/ERF collinear gene pairs were identified. Then, Ka, Ks, and Ka/Ks ratios of these 298 AP2/ERF collinear gene pairs were calculated to estimate the divergence time (T value) and selection pressure among duplicated SmAP2/ERF gene pairs (Table S5). All of the Ka/Ks values were below 1, which indicated that these genes might have experienced strong purifying selective pressure during evolution. Among the 298 AP2/ERF collinear gene pairs, 198 were located on duplicated segments on 38 chromosomes in Salix matsudana (Fig. 5 and Table S4). The collinear gene pairs in the Salix matsudana genome were visualized by Circos, and the gene pairs were linked by lines (grey lines indicated all gene pairs, red lines indicated AP2/ERF collinear gene pairs. Synteny analysis of AP2/ERF genes between Salix matsudana and two related Salicaceae species , Populus trichocarpa and Salix purpurea To further infer the phylogenetic mechanisms of the SmAP2/ERF family, we constructed two comparative syntenic maps of Salix matsudana with two related species, Populus trichocarpa and Salix purpurea (Fig. 6 ).Collinear AP2/ERF genes pairs between Salix matsudana and other two species are listed in Supplementary Table S6. A total of 263 SmAP2/ERF genes showed syntenic relationships with 183 genes from Populus trichocarpa , and 248 SmAP2/ERF genes showed syntenic relationships with 144 genes from Salix purpurea . The number of orthologous pairs between Salix matsudana and Populus trichocarpa , and Salix matsudana and Salix purpurea were 423 and 292, respectively (Supplementary Table S6). Some PtAP2/ERF and SpAP2/ERF genes were found to be associated with at least four syntenic gene pairs. Interestingly, the number of collinear gene pairs identified between Salix matsudana and Salix purpurea were less than that between Salix matsudana and Populus trichocarpa . In the comparative syntenic map between Salix matsudana and Populus trichocarpa , syntenic links were found between all 19 Populus trichocarpa chromosomes and all 38 Salix matsudana chromosomes. Alternatively, in the comparative syntenic map between Salix matsudana and Salix purpurea , there were no syntenic links between Chr1, Chr12, and Chr36 from Salix matsudana , and Chr15Z and Chr15W from Salix purpurea . Specific Expression Of AP2/ERF Superfamily Genes Under Salt Stress To investigate the physiological roles of SmAP2/ERF genes in salt stress tolerance, we identified the expression patterns of SmAP2, SmERF, and SmDREB subgroup genes from the RNA sequencing data. By inputting the FPKM values (Fragments Per Kilobase of transcript per Million fragments mapped) of these genes in TBtools, three heatmaps were constructed to demonstrate the expression pattern change under salt stress (Fig. 7 ). The expression patterns of 286 genes are illustrated in Fig. 7 , and included 48 AP2, 108 DREB, and 130 ERF subgroup genes. In the AP2 subgroup, the FPKM values of 31 genes were < 3, which indicated lower expression in the root and no response to salt stress. Five genes had differential expression patterns. The expression levels of four genes ( SmAP2-38 , SmAP2-4 , SmAP2-3 , and SmAP2-33 ) were induced by salt stress, whereas the expression of gene SmAP2-15 decreased after salt stress. In the DREB subgroup, 108 genes were present in the heatmap, and expression levels of 10 genes, such as SmDREB A1-10 , SmDREB A1-9 , and SmDREB A1-7 , were induced by salt stress and remained higher. In the ERF subgroup heatmap, 130 genes were included. The expression levels of 13 genes were upregulated by salt stress, but only the expression of SmERF B4-1 was higher. Three genes were downregulated by salt stress, including SmERF B3-52 . In many paralog gene pairs, we found one gene with higher expression, whereas the other gene had lower expression, such as SmDREB-A9 / SmDREB-A10 , SmAP2-33 / SmAP2-39 and SmERF-9 / SmERF-10 gene pairs. Thirteen Genes with upregulated expression patterns was verified by qRT-PCR (Real-time Quantitative PCR) (Fig. 8 ). Discussion Salix is one of the few woody plants with a large number of polyploid taxa, in Salix matsudana , both tetraploid and diploid individuals have been observed. [ 25 ]. In our experiment, we sequenced the genome of tetraploid Salix matsudana . Tetraploid Salix have value because they have higher tolerance to abiotic stress than their diploid relatives; therefore, they can be planted beachside to alleviate soil salinity and improve the ecological environment [ 22 ]. The molecular mechanism of salinity response regulation is very complex, and AP2/ERF TFs are key regulators in plants [ 26 ]. Here, we identified 364 AP2/ERF gene members in Salix matsudana , and characterized their classification, chromosome location, gene structure, and syntenic relationships of these genes within the genome and between other species. We also revealed the expression patterns under salt stress. These efforts can serve as a first step in comprehensive functional characterization of AP2/ERF genes by reverse genetic approaches and molecular genetics research. As a tetraploid species, Salix matsudana has more AP2/ERF gene members than other plants selected for comparison, including three Salicaceae family relatives (Table S2). The total number of genes is approximately double compared with poplar and two willow relatives, but the proportions of some subgroups were slightly different. Salix arbutifolia had a higher percentage (50.8% > 45%) of ERF subfamily members, but a lower proportion (33% < 38%) of DREB subfamily members compared with other species (Table S2). For DREB-A1 and ERF-B2 subgroups, the highest percentage was found in Salix purpurea , and there were the same number of or more members of these two subgroups compared with other species, including the tetraploid Salix matsudana . For ERF-B3, Salix arbutifolia had the highest subgroup percentage (18.5%). In Salix matsudana , ERF-B6 had the lowest percentage (5.7%), whereas the subfamily AP2 had the highest percentage (15.1%). These data indicated that, during evolution, AP2/ERF family subgroup members probably underwent gene duplication or loss and therefore evolved into the specific AP2/ERF subgroup proportions in each species. A phylogenetic tree that included 364 genes from Salix matsudana and 48 genes from A. thaliana and Populus trichocarpa was constructed (Fig. 1 ). All subgroups were clustered together. Eight genes with one AP2 domain were classified into the AP2 family because of a close phylogenetic relationship. This classification was similar to that in Arabidopsis , in which four genes involved in the AP2 family contained a single AP2 domain [ 1 ]. The gene intron/exon structure and conserved motifs were identified in the 364 SmAP2/ERF members. Similar to that of the AP2/ERF genes from other species, such as cauliflower and radish, the AP2 subfamily had more introns and the ERF subfamily had fewer [ 18 , 19 ]. Previous studies found that intron number and distribution are related to plant evolution, and introns of the ERF family genes were probably lost during evolution in higher plants [ 27 , 28 ]. In total, 215 of the 301 members (70%) of the ERF family had no introns, which was a little less than that of tartary buckwheat [ 24 ] and also consistent with previous findings. Through the conserved domains and motifs, TFs play roles in gene expression regulation by promoter binding, transcription activation, and protein–protein interactions [ 29 ]. Motif analysis showed that Motif-6, Motif-8, and Motif-10 were specifically detected in different groups of the AP2/ERF subfamily; seven other motifs were all related to the AP2 domain (Fig. 3 b). Motif-8 was specifically found in the DREB subgroup, such as in the DREB-A1, DREB-A4, and DREB-A5 clades. Motif-10 was mostly found distributed on proteins from the ERF-B3, DREB-A2, and DREB-A4 clades. Motif-6 was specifically located between the two AP2 domains of AP2 subgroup members. These results indicate that, although some motifs of the AP2/ERF family genes were highly conserved and involved in DNA binding, such as motifs from the AP2 domain, the functions of other subgroup-specific motifs are still unknown, and more work is required to clarify their regulatory functions. Based on the genome assembly data, 301 genes were anchored on the 38 chromosomes (LGs), but they were unevenly distributed. Eleven TDs were found on 11 chromosomes, and seven tandem duplication gene pairs came from the SmERF B3 subgroup, which included three duplicated genes ( SmERF B3-6 , SmERF B3-7 , and SmERF B3-8 ) that clustered together. Apart from the tandem duplication cluster, SmERF B3 members typically clustered on a chromosome, with three genes as a unit. In 12 clusters, 37 SmERF B3 genes were found. This phenomena were also found in Populus trichocarpa , thirteen PtERF B3 genes located in 4 clusters, which indicated that in the evolution of Sm, apart from the chromosome duplication, segmental duplication were also happened. Using MCScanX, we found a total of 28,348 collinear gene pairs in the Salix matsudana genome, from which 299 AP2/ERF collinear gene pairs were identified; this indicated that, during evolution, the Salix matsudana genome experienced a whole genome duplication event. Population genetic theory predicts that, after duplication, some redundant duplicate copies will be silenced and eliminated, and other retained paralogs will obtain sub- or neofunction by DNA mutation in coding or regulatory sequences [ 30 – 32 ]. Then, we calculated Ka, Ks, and Ka/Ks ratios of these 298 AP2/ERF collinear gene pairs to estimate the divergence time and selection pressure. All Ka/Ks values were below 1, which indicated that these genes might have experienced strong purifying selective pressure during evolution. It was previously reported that purifying selection would lead to the loss of redundant genes [ 33 ]. Based on the gene number of most subgroups, we did not find any obvious evidence of gene loss, but in the RAV subgroup, there was an exception; there were only six members in Salix matsudana , which is identical to the gene number in Arabidopsis . Based on the gene loss hypothesis, the duplication paralogs of RAVs may have been lost during genome evolution because of their rapid evolutionary rate. Approximately 52–59 million years ago (Mya), willow and poplar, which are two modern taxa, originated from a diploid progenitor, but when and how Salix matsudana experienced chromosome duplication remains largely unknown [ 34 ]. The divergence time (T Value) of gene pairs mainly occurred during two time periods, 2–8 and 20–36 Mya. Gene pairs with a divergence time of 20–36 Mya were probably paralogs before whole genome duplication events, whereas 2–8 Mya is probably the divergence time of paralogs after whole genome duplication events. These data indicated that the progenitor of Salix matsudana underwent whole genome duplication not more than 10 Mya. Similar to the findings of a previous report [ 35 ], alignment of a Salix linkage map to the Populus genomic sequence revealed macrosynteny between willow and poplar genomes (Fig. 6 A, 6 B). Synteny analysis of Salix matsudana vs Populus trichocarpa , and Salix matsudana vs Salix purpurea revealed 423 and 292 orthologous pairs, respectively. In total, 263 SmAP2/ERF genes had syntenic relationships with 183 genes in Populus trichocarpa , whereas 248 SmAP2/ERF genes showed syntenic relationships with 144 genes in Salix purpurea . Interestingly, the collinear gene pairs identified between Salix matsudana and Salix purpurea were less than that from Salix matsudana and Populus trichocarpa . Syntenic links were found between all 19 Populus trichocarpa chromosomes and all 38 Salix matsudana chromosomes, but there were no syntenic links between Chr1, Chr12, and Chr36 from Salix matsudana and Chr15Z and Chr15W from Salix purpurea . Salix has 300–500 species and considerable variation, ranging from shrubs to trees [ 36 ]; willow may evolve faster, which would lead to them being more diverse. Researchers proposed that Populus might be evolutionarily more primitive than Salix [ 30 , 37 ]. From our results, we could infer the evolutionary relationships of three Salicaceae species ( Populus trichocarpa , Salix matsudana , and Salix purpurea ); Populus trichocarpa was the most primitive taxon, Salix purpurea was the most derived taxon, and Salix matsudana was located between them but was genetically more closely related to Populus trichocarpa than Salix purpurea . Plants must adapt to various biotic and abiotic stresses because they are immobile in their life cycles. For example, Salix matsudana must adapt to the soil salinity when grown along coastal beaches. Consequently, some AP2/ERF TFs play important roles in plants by facilitating defense against stress and improving resistance. From the RNA sequencing data, we extracted the expression FPKM values and constructed expression heatmaps to show the expression patterns under salt stress (Fig. 7 ). The expression levels of four genes from the AP2 subgroup, 10 genes from the DREB subgroup, and 13 genes from the ERF subgroup were induced by salt stress, but only the expression levels of four genes were downregulated after salt stress. The expression patterns were verified by qRT-PCR. The expression pattern of many AP2/ERF gene pairs with evolutionary relationships differed, which indicated that the AP2/ERF gene family may have changed at the transcriptional regulation level followi polyploidization. That finding provides additional evidence that redundant duplicated gene pairs experienced functional divergence based on expression pattern change. These differentially expressed SmAP2/ERF genes could be selected as candidate genes; further exploration on their roles under salt stress will reveal molecular mechanisms responsible for salinity stress responses in Salix matsudana . In conclusion, 364 AP2/ERF TFs were identified in Salix matsudana . Clustering and phylogenetic analysis were conducted to classify these TFs into 15 subgroups. Chromosome location, gene structure, and conserved motifs were identified for 364 AP2/ERF TFs. Evolutionary relationships of these genes were revealed by tandem and segmental duplication gene pair identification, divergence time estimation, and T value calculation, which indicated that the progenitor of Salix matsudana underwent whole genome duplication not more than 10 Mya. Synteny analysis with other species showed macrosynteny between willow and poplar AP2/ERF genes, and Salix matsudana was genetically more closely related to Populus trichocarpa than Salix purpurea . The AP2/ERF TFs were also confirmed to exhibit differential expression patterns during salt stress. The functions of these genes should be investigated in future studies to better clarify the mechanism of salt tolerance regulation in Salix matsudana . Conclusion In this study, 364 SmAP2/ERF genes of Salix matsudana were identified and renamed according to the chromosomal location of the SmAP2/ERF genes. Gene classification, gene structure and conserved motifs were analyzed in detail. Investigation results on syntenic relationships between the SmAP2/ERF genes and AP2/ERF genes from other species elucidated that the progenitors of Salix matsudana underwent whole genome duplication not more than 10 Mya and Salix matsudana is genetically more closely related to Populus trichocarpa than to Salix purpurea . Moreover, analyses on the differential expression patterns of SmAP2/ERF genes during salt stress can help to reveal the mechanism of salt tolerance regulation in Salix matsudana . Methods Material and salt stress treatment The salinity stress treatments were carried out on Salix matsudana ‘yanjiang’ and Salix matsudana ‘9901’. The 2 salix cultivars used in this study were collected from the botany garden of Nantong University (Nantong, China). The 2 salix cultivars were authorized for only scientific research purpose, and were deposited in College of life science in Nantong University. The stem cuttings (length, 8–10 cm; coarse, 2–3 cm) of two materials were selected for hydroponic rooting for 20 days and then treated with NaCl solution (150 mM) for 4 h. All root samples were divided into four categories with three biological replicates: Sample1-1/Sample1-2/Sample1-3 (‘yanjiang’ without salt stress treatment), Sample1N-1/Sample1N-2/Sample1N-3 (‘yanjiang’ with salt stress treatment), Sample2-1/Sample2-2/Sample2-3 (‘9901’ without salt stress treatment), and Sample2N-1/Sample2N-2/Sample2N-3 (‘9901’ with salt stress treatment). Genome Sequence Retrieval The Populus trichocarpa and Salix purpurea sequences were downloaded from JGI ( http://www.phytozome.net/ ). The Salix matsudana sequences were obtained from our sequencing, and assembly results were obtained by Roche/454 and Illumina/HiSeq-2000 sequencing technologies (unpublished). Identification of AP2/ERF genes in Salix matsudana and Salix purpurea The Pfam accession number of AP2 domain is PF00847.16. We downloaded the Hidden Markov Model (HMM) profile for the AP2/ERF TFs from the Pfam database ( http://pfam.xfam.org/ ) with Pfam accession number PF00847.16 as the search keyword. An alternative HMM profile was built by sequence alignment using Clustal W[ 38 ]. Using an in-house Perl script with two HMM profiles as queries, hmmsearch was carried out by searching the Salix matsudana and Salix purpurea protein databases with default parameters. To validate the putative accuracy of two HMM search results, the candidate protein sequences were checked in three websites: SMART ( http://smart.embl.de/# ), CDD ( https://www.ncbi.nlm.nih.gov/cdd/ ), and Pfam ( http://pfam.xfam.org/ ). Candidate proteins with positive results from all three websites were selected as AP2/ERF family members of Salix matsudana and Salix purpurea . Additionally, putative AP2/ERF protein characteristics, including length, molecular weight, and isoelectric point, were calculated by the ExPasy site ( http://au.expasy.org/tools/pi_tool.html ). Phylogenetic Analysis And Classification Of AP2/ERF Genes Using an in-house Perl script (domain_xulie.pl), the conserved AP2 core domains of putative SmAP2 proteins were obtained and subjected to multiple sequence alignment using ClustalW [ 38 ]. To better classify these SmAP2 genes, 48 AP2 domains from known categories of Arabidopsis and Populus trichocarpa AP2 genes were selected to carry out multiple sequence alignment with SmAP2 proteins, and a phylogenetic tree based on this alignment was built by MEGA 7.0 with the neighbor-joining method [ 39 ] with default parameters. Bootstrap value was set to 1000. Depending on the phylogenetic tree constructed by SmAP2, PtAP2, and AtAP2 domains, these SmAP2 genes were classified into different subfamilies and subgroups. Gene Structure And Conserved Motif Structure Analysis The UTR–exon–intron structures of the SmAP2 genes were obtained based on the gene annotation gff3 files we assembled. Using the online website tool Gene Structure Display Server ( http://gsds.cbi.pku.edu.cn/ ), we obtained the gene structure diagrams [ 40 ]. To characterize the SmAP2 protein structure, the online tool MEME ( http://meme-suite.org/tools/meme ) was used to search for conserved motifs [ 41 ]. The optimized parameters were employed as follows: any number of repetitions, maximum number of motifs = 10, and the optimum width of each motif was 6–50 residues. The search result file meme.xml was downloaded from the website and opened by TBtools v0.66831 to obtain the gene structure diagram [ 42 ]. Gene position on chromosomes, and gene tandem and segmental duplication analysis Using the “Amazing Gene Location From GFF3/GTF File” tool of TBtools, the SmAP2 genes were mapped on 38 chromosomes of Salix matsudana . Because some scaffolds were not assembled onto the chromosomes, not all SmAP2 genes mapped onto the chromosomes. Salix matsudana is a tetraploid willow. Gene duplication events are a common phenomenon in the genome. There are two kinds of gene duplications in the genome: tandem duplication events (TDs) and segmental duplication events (SDs). TDs refer to two or more adjacent homologous genes located within 200 Kb on a single chromosome; SDs refer to homologous gene pairs between different chromosomes [ 43 ]. The gene duplication pairs were identified in TBtools by the “Blast compare 2 Seq [sets] ” and “Quick McscanX Wrapper” tools. The candidate duplicated genes should have ≥ 80% coverage and ≥ 65% similarity. The TDs of SmAP2 genes were revealed on a chromosome using the “Amazing Gene Location From GFF3/GTF File” tool of TBtools. The SDs of SmAP2 genes were visualized by the “Amazing Super Circos” tool of TBtools. Divergence Time Calculation Of Duplicated Genes After BLASTn analysis of Coding sequences and obtaining duplicated gene pairs, the nonsynonymous (Ka) and synonymous (Ks) pairs were calculated by the “Simple Ka/Ks calculator (NG)” tool of TBtools. The divergence time was estimated with the formula: T = Ks/2λ. The clock-like rate λ value (9.1 × 10 − 9 ) from Populus was used in the calculation. [ 44 ] Collinearity analysis between Salix matsudana and the representative species To demonstrate the syntenic relationships of the orthologous AP2 genes obtained from Salix matsudana and other two selected plants ( Populus trichocarpa , and Salix purpurea ,), the syntenic analysis maps were constructed using the “Amazing Super Circos” tool of TBtools. RNA sequencing and a heat map generated by hierarchical clustering Transcriptome sequencing data of 12 samples were obtained by Illumina HiSeq sequencing. The expression levels of genes in different samples were calculated using FPKM values. Using the “Amazing HeatMap” tool of TBtools, a graph of the expression level of AP2 family genes with hierarchical clustering was generated. RNA Isolation And qRT-PCR Analysis Total RNA was extracted using TaKaRa MiniBEST Plant RNA Extraction Kit (Takara, Dalian, China) from roots according to the manufacturer’s instruction. For each sample, 3 µg of total RNA was used to synthesize first-strand cDNA with SuperScriptII reverse transcriptase (Takara, Dalian, China). For qRT-PCR, the reaction preparation, application parameter settings and quantitative analysis were performed as previously described [ 45 ]. The reactions were performed using the ABI Prism 7000 Real-time PCR system (Applied Biosystems, USA). The Salix purpurea Actin1 gene (SapurV1A.0655s0050.1) were used as reference genes. The gene-specific primers for the 13 selected genes are listed in Table S7. Abbreviations AP2/ERF AP2-like ethylene-responsive transcription factor FPKM Fragments Per Kilobase of transcript per Million fragments mapped qRT-PCR Real-time Quantitative PCR Mya million years ago HMM Hidden Markov Model TDs tandem duplication events SDs segmental duplication events TFs transcription factors Declarations Ethics approval and consent to participate Not applicable . Consent for publication Not applicable. Availability of data and materials The Salix matsudana genomic sequence datasets used during the current study are available from the corresponding author on reasonable request. Other data generated or analysed during this study are included in this published article and its supplementary information files. Competing Interests The authors declare that they have no conflict of interest. Funding This work was supported by the National Natural Science Foundation of China (31971681), Jiangsu Province Forestry Science and Technology Innovation and Promotion Project (LYKJ [2018]36) and Nantong University Scientific Research Start-up project for Introducing Talents (18R08). The funding body has no role in the study design and data collection, analysis, interpretation of data and in writing the manuscript. Authors' contributions Y.C. and J.Z. conceived the research plans; Y.C. and Y.J. performed most of the experiments; G.L., C.Y., B.L., F.Z. and W.F. performed the rest of the experiments; Y.C. analyzed the data; Y.C. and J.Z. wrote the article. All authors have read and approved the manuscript. Acknowledgments We thank Mallory Eckstut, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. References Nakano T, Suzuki K, Fujimura T, Shinshi H: Genome-wide analysis of the ERF gene family in Arabidopsis and rice . Plant Physiol 2006, 140 (2):411-432. Song X, Wang J, Ma X, Li Y, Lei T, Wang L, Ge W, Guo D, Wang Z, Li C et al : Origination, Expansion, Evolutionary Trajectory, and Expression Bias of AP2/ERF Superfamily in Brassica napus . Front Plant Sci 2016, 7 :1186. Zhuang J, Cai B, Peng RH, Zhu B, Jin XF, Xue Y, Gao F, Fu XY, Tian YS, Zhao W et al : Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa . Biochem Biophys Res Commun 2008, 371 (3):468-474. Gu C, Guo ZH, Hao PP, Wang GM, Jin ZM, Zhang SL: Multiple regulatory roles of AP2/ERF transcription factor in angiosperm . Botanical studies 2017, 58 (1):6. Duran-Medina Y, Serwatowska J, Reyes-Olalde JI, de Folter S, Marsch-Martinez N: The AP2/ERF Transcription Factor DRNL Modulates Gynoecium Development and Affects Its Response to Cytokinin . Front Plant Sci 2017, 8 :1841. Wessels B, Seyfferth C, Escamez S, Vain T, Antos K, Vahala J, Delhomme N, Kangasjarvi J, Eder M, Felten J et al : An AP2/ERF transcription factor ERF139 coordinates xylem cell expansion and secondary cell wall deposition . New Phytol 2019. Gao Y, Liu Y, Liang Y, Lu J, Jiang C, Fei Z, Jiang CZ, Ma C, Gao J: Rosa hybrida RhERF1 and RhERF4 mediate ethylene- and auxin-regulated petal abscission by influencing pectin degradation . Plant J 2019, 99 (6):1159-1171. Li H, Xiao Q, Zhang C, Du J, Li X, Huang H, Wei B, Li Y, Yu G, Liu H et al : Identification and characterization of transcription factor ZmEREB94 involved in starch synthesis in maize . J Plant Physiol 2017, 216 :11-16. Shen SL, Yin XR, Zhang B, Xie XL, Jiang Q, Grierson D, Chen KS: CitAP2.10 activation of the terpene synthase CsTPS1 is associated with the synthesis of (+)-valencene in 'Newhall' orange . J Exp Bot 2016, 67 (14):4105-4115. Srivastava R, Kumar R: The expanding roles of APETALA2/Ethylene Responsive Factors and their potential applications in crop improvement . Briefings in functional genomics 2018, 18 (4):240-254. Li Y, Zhang H, Zhang Q, Liu Q, Zhai H, Zhao N, He S: An AP2/ERF gene, IbRAP2-12, from sweetpotato is involved in salt and drought tolerance in transgenic Arabidopsis . Plant Sci 2019, 281 :19-30. Cao B, Shu L, Li A: Functional characterization of LkERF-B2 for improved salt tolerance ability in Arabidopsis thaliana . 3 Biotech 2019, 9 (7):263. Abogadallah GM, Nada RM, Malinowski R, Quick P: Overexpression of HARDY , an AP2/ERF gene from Arabidopsis, improves drought and salt tolerance by reducing transpiration and sodium uptake in transgenic Trifolium alexandrinum L . Planta 2011, 233 (6):1265-1276. Jin T, Chang Q, Li W, Yin D, Li Z, Wang D, Bao L, Liu L: Stress-inducible expression of GmDREB1 conferred salt tolerance in transgenic alfalfa . Plant Cell Tissue & Organ Culture 2010, 100 (2):219-227. Brendel V, Kurtz S, Walbot V: Comparative genomics of Arabidopsis and maize: prospects and limitations . Genome Biol 2002, 3 (3):. reviews1005.1. Hall AE, Fiebig A, Preuss D: Beyond the Arabidopsis genome: opportunities for comparative genomics . Plant Physiol 2002, 129 (4):1439-1447. Schranz ME, Song BH, Windsor AJ, Mitchell-Olds T: Comparative genomics in the Brassicaceae: a family-wide perspective . Curr Opin Plant Biol 2007, 10 (2):168-175. Karanja BK, Xu L, Wang Y, Tang M, M'Mbone Muleke E, Dong J, Liu L: Genome-wide characterization of the AP2/ERF gene family in radish (Raphanus sativus L.): Unveiling evolution and patterns in response to abiotic stresses . Gene 2019, 718 :144048. Li H, Wang Y, Wu M, Li L, Li C, Han Z, Yuan J, Chen C, Song W, Wang C: Genome-Wide Identification of AP2/ERF Transcription Factors in Cauliflower and Expression Profiling of the ERF Family under Salt and Drought Stresses . Front Plant Sci 2017, 8 :946. Li MY, Liu JX, Hao JN, Feng K, Duan AQ, Yang QQ, Xu ZS, Xiong AS: Genomic identification of AP2/ERF transcription factors and functional characterization of two cold resistance-related AP2/ERF genes in celery (Apium graveolens L.) . Planta 2019, 250 (4):1265-1280. Zhang J, Yuan H, Yang Q, Li M, Wang Y, Li Y, Ma X, Tan F, Wu R: The genetic architecture of growth traits in Salix matsudana under salt stress . Hortic Res 2017, 4 :17024. Zhang J, Yuan H, Li M, Li Y, Wang Y, Ma X, Zhang Y, Tan F, Wu R: A High-Density Genetic Map of Tetraploid Salix matsudana Using Specific Length Amplified Fragment Sequencing (SLAF-seq) . PLoS One 2016, 11 (6):e0157777. Rao G, Sui J, Zeng Y, He C, Zhang J: Genome-wide analysis of the AP2/ERF gene family in Salix arbutifolia . FEBS open bio 2015, 5 :132-137. Liu M, Sun W, Ma Z, Zheng T, Huang L, Wu Q, Zhao G, Tang Z, Bu T, Li C et al : Genome-wide investigation of the AP2/ERF gene family in tartary buckwheat (Fagopyum Tataricum) . BMC Plant Biol 2019, 19 (1):84. Guo W, Hou J, Yin T, Chen Y: An analytical toolkit for polyploid willow discrimination . Scientific reports 2016, 6 :37702-37702. Xie Z, Nolan TM, Jiang H, Yin Y: AP2/ERF Transcription Factor Regulatory Networks in Hormone and Abiotic Stress Responses in Arabidopsis . Front Plant Sci 2019, 10 :228. Tang Y, Qin S, Guo Y, Chen Y, Wu P, Chen Y, Li M, Jiang H, Wu G: Genome-Wide Analysis of the AP2/ERF Gene Family in Physic Nut and Overexpression of the JcERF011 Gene in Rice Increased Its Sensitivity to Salinity Stress . Plos One 2016, 11 (3):e0150879. Zhang Z, Li X: Genome-wide identification of AP2/ERF superfamily genes and their expression during fruit ripening of Chinese jujube . Scientific reports 2018, 8 (1):15612. Liu L, ., White MJ, Macrae TH: Transcription factors and their genes in higher plants functional domains, evolution and regulation . Febs Journal 1999, 262 (2):247-257. Hou J, Wei S, Pan H, Zhuge Q, Yin T: Uneven selection pressure accelerating divergence of Populus and Salix . Horticulture Research 2019, 6 (1):37. Xionglei H, Jianzhi Z: Rapid subfunctionalization accompanied by prolonged and substantial neofunctionalization in duplicate gene evolution . Genetics 2005, 169 (2):1157-1164. Adams KL, Richard C, Ryan P, Wendel JF: Genes duplicated by polyploidy show unequal contributions to the transcriptome and organ-specific reciprocal silencing . P Natl Acad Sci USA 2003, 100 (8):4649-4654. Kondrashov FA, Rogozin IB, Wolf YI, Koonin EV: Selection in the evolution of gene duplications . Genome Biology 2002, 3 (2):1-9. Hou J, Ye N, Dong Z, Lu M, Li L, Yin T: Major Chromosomal Rearrangements Distinguish Willow and Poplar After the Ancestral “Salicoid” Genome Duplication . Genome Biology & Evolution 2016, 8 (6):1868-1875. Hanley SJ, Mallott MD, Karp A: Alignment of a Salix linkage map to the Populus genomic sequence reveals macrosynteny between willow and poplar genomes . Tree Genetics & Genomes 2006, 3 (1):35-48. Argus GW: Infrageneric Classification of Salix (Salicaceae) in the New World . Systematic Botany Monographs 1997, 52 :1-121. Dai X, Hu Q, Cai Q, Kai F, Ning Y, Tuskan GA, Milne R, Chen Y, Wan Z, Wang Z: The willow genome and divergent evolution from poplar after the common genome duplication . Cell Research 2014, 24 (10):1274-1277. Larkin M, Blackshields G, Brown N, Chenna R, Mcgettigan P, Mcwilliam H, Valentin F, Wallace I, Wilm A, R: Clustal W and clustal X version 2.0 . Bioinformatics 2007, 23 (21):2947-2948. Kumar S, Stecher G, Tamura K: MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets . Molecular Biology & Evolution 2016, 33 (7):1870. Hu B, Jin J, Guo AY, Zhang H, Luo J, Gao G: GSDS 2.0: an upgraded gene feature visualization server . Bioinformatics 2014, 31 (8):1296. Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS: MEME SUITE: tools for motif discovery and searching . Nucleic Acids Res 2009, 37 (Web Server issue):W202-208. Chen C, Xia R, Chen H, He Y: TBtools, a Toolkit for Biologists integrating various HTS-data handling tools with a user-friendly interface . bioRxiv 2018:doi: 10.1101/289660. Cannon SB, Mitra A, Baumgarten A, Young ND, May G: The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana . BMC Plant Biology 2004, 4 (1):10. Lynch M, Conery JS: The evolutionary fate and consequences of duplicate genes . Science 2000, 290 (5494):1151-1155. Chen YH, Cao YY, Wang LJ, Li LM, Yang J, Zou MX: Identification of MYB transcription factor genes and their expression during abiotic stresses in maize . Biol Plantarum 2018, 62 (2):222-230. Supplementary Files SupplementalfilesTableS1S7FigureS1S2.docx Cite Share Download PDF Status: Published Journal Publication published 13 Apr, 2021 Read the published version in PeerJ → Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-15225","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":371132,"identity":"a9e96c64-e50f-4d36-84d5-63a4c9a7c78a","order_by":1,"name":"Yanhong Chen","email":"","orcid":"","institution":"Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Yanhong","middleName":"","lastName":"Chen","suffix":""},{"id":371133,"identity":"ff2409dc-61c4-44e6-a551-37b862bc8b6c","order_by":2,"name":"Yuna Jiang","email":"","orcid":"","institution":"Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Yuna","middleName":"","lastName":"Jiang","suffix":""},{"id":371134,"identity":"60107893-a4ab-471e-b460-2050824951b4","order_by":3,"name":"Guoyuan Liu","email":"","orcid":"","institution":"Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Guoyuan","middleName":"","lastName":"Liu","suffix":""},{"id":371135,"identity":"3237ebe5-3494-4100-a322-89ee3fde6853","order_by":4,"name":"Chunmei Yu","email":"","orcid":"","institution":"Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Chunmei","middleName":"","lastName":"Yu","suffix":""},{"id":371136,"identity":"ac03557e-bd66-47aa-b0ea-3324c246c2ea","order_by":5,"name":"Bolin Lian","email":"","orcid":"","institution":"Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Bolin","middleName":"","lastName":"Lian","suffix":""},{"id":371137,"identity":"67757972-d84c-4290-a26e-72dabf630357","order_by":6,"name":"Fei Zhong","email":"","orcid":"","institution":"Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Zhong","suffix":""},{"id":371138,"identity":"8c5c2dbb-fd04-4220-89f3-5b5d4107fbaf","order_by":7,"name":"Wenxiang Feng","email":"","orcid":"","institution":"Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Wenxiang","middleName":"","lastName":"Feng","suffix":""},{"id":371139,"identity":"e3e67523-148d-4f57-b4bb-ac93b87c2b74","order_by":8,"name":"Jian Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYHACAwaGAgYGfmbmww+I08AG0gJEku1saQakaTE4z6MgQZQOc/nmbdI8BnWJmw/zAHXW2EQT1GLZxlZszGPAlrjtMO+BBwzH0nIbCGkxOMZj+JjHgCd322G+BAPGhsNEaTE4zGMgkbu5GUgSqwVki0HuBmbitaQVG84xSKifcRgYyAlE+eXw4W0SbyrqjPn7Dx9+8KHGhrAWEGDigbESiFEOAow/iFU5CkbBKBgFIxMAAFVTOdoF/IypAAAAAElFTkSuQmCC","orcid":"","institution":"Nantong University","correspondingAuthor":true,"prefix":"","firstName":"Jian","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2020-02-25 13:36:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.2.24667/v1","doiUrl":"https://doi.org/10.21203/rs.2.24667/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.7717/peerj.11076","type":"published","date":"2021-04-13T21:05:49+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":554834,"identity":"2b5aa931-fa30-43e2-a41f-19b73ad2065d","added_by":"auto","created_at":"2020-02-27 02:21:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":404331,"visible":true,"origin":"","legend":"Unrooted phylogenetic tree and classification of 364 SmAP2/ERF genes and their representative orthologs from Arabidopsis and Populus.\nThe amino acid sequences of AP2 domains from 364 SmAP2/ERF proteins and 48 orthologs from Arabidopsis and Populus were aligned by ClustalW, and the neighbor-joining tree was constructed using MEGA 7.0 with 1000 bootstrap replicates. The evolutionary distances were computed using the p-distance method. In total, 364 SmAP2/ERF members were classified into 15 smaller subgroups, and their names are labeled beside the tree.","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/0f5e8f94-007b-4a68-8ffc-04ca646c5fa4/v1/fig1.png"},{"id":554835,"identity":"92c772b8-58e5-4323-b05d-e6b2fbcab4c6","added_by":"auto","created_at":"2020-02-27 02:21:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":128112,"visible":true,"origin":"","legend":"Classification and subgroup proportions of SmAP2/ERF family genes. \nThe size of each piece is proportional to the relative abundance to the SmAP2/ERF genes assigned to this group.","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/0f5e8f94-007b-4a68-8ffc-04ca646c5fa4/v1/fig2.png"},{"id":554836,"identity":"60977c49-693d-4d4b-9447-a0e36e1fc820","added_by":"auto","created_at":"2020-02-27 02:21:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":230098,"visible":true,"origin":"","legend":"Phylogenetic relationships, gene structure, and architecture of conserved protein motifs in SmAP2/ERF superfamily memebers.\na, The phylogenetic tree was constructed based on the amino acid sequences of the AP2 domain from 364 SmAP2/ERF proteins using MEGA7.0. b, Motif composition of tartary buckwheat AP2/ERF proteins. Motifs 1–10 are displayed in different colored boxes. The sequence information for each motif is provided in Figure S2. c, Exon/intron structure of SmAP2/ERF genes. Yellow boxes indicate untranslated 5′- and 3′-regions; green boxes indicate exons; black lines indicate introns. The protein length can be estimated using the scale at the bottom.","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/0f5e8f94-007b-4a68-8ffc-04ca646c5fa4/v1/fig3.png"},{"id":554837,"identity":"7b70efa9-4e7d-45f8-998f-ad188fdde7e4","added_by":"auto","created_at":"2020-02-27 02:21:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":431167,"visible":true,"origin":"","legend":"Schematic representations for the chromosomal distribution and tandem duplication of SmAP2/ERF genes. \nThe red lines indicate tandem duplicated AP2/ERF gene pairs, which are indicated in green. The SmERF-B3 subgroup members labeled with blue clustered on the same chromosome. The chromosome number is indicated to the left of each chromosome.","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/0f5e8f94-007b-4a68-8ffc-04ca646c5fa4/v1/fig4.png"},{"id":554838,"identity":"c1eccbdb-dd64-4d95-8878-b320755a8b15","added_by":"auto","created_at":"2020-02-27 02:21:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":332407,"visible":true,"origin":"","legend":"Schematic representations of the segmental duplication and interchromosomal relationships of SmAP2/ERF genes. \nGrey lines indicate all syntenic gene pairs in the Salix matsudana genome, red lines indicate syntenic relationships between SmAP2/ERF genes. The orange color columes outside of the circle indicated the gene density on each 38 chromosomes. The deeper color means the higher density of genes.","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/0f5e8f94-007b-4a68-8ffc-04ca646c5fa4/v1/fig5.png"},{"id":554839,"identity":"142e97fc-4b70-4d11-9ff2-19be6187d0ef","added_by":"auto","created_at":"2020-02-27 02:21:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":626025,"visible":true,"origin":"","legend":"Synteny analysis of AP2/ERF genes between Salix matsudana and two related Salicaceae species, Populus trichocarpa and Salix purpurea. \na, Synteny analysis of AP2 genes between Salix matsudana and Populus trichocarpa. b. Synteny analysis of AP2 genes between Salix matsudana and Salix purpurea\nGray lines in the background indicate the collinear blocks within Salix matsudana and other plant genomes, whereas red lines highlight syntenic AP2/ERF gene pairs.","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/0f5e8f94-007b-4a68-8ffc-04ca646c5fa4/v1/fig6.png"},{"id":554840,"identity":"dc78a17f-9e49-4b7e-aa3d-45efd4f14be7","added_by":"auto","created_at":"2020-02-27 02:21:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":651397,"visible":true,"origin":"","legend":"Hierarchical clustering of AP2 genes and heatmap that demonstrates the differential expression patterns of SmAP2/ERF genes in roots before and after salt stress.\na, Heatmap and hierarchical clustering representation of 48 AP2 members. b, Heatmap and hierarchical clustering representation of 108 DREB members. c, Heatmap and hierarchical clustering representation of 130 ERF members.","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/0f5e8f94-007b-4a68-8ffc-04ca646c5fa4/v1/fig7.png"},{"id":554841,"identity":"46d972a1-a426-42f5-a27f-d11be84d72e0","added_by":"auto","created_at":"2020-02-27 02:21:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":99752,"visible":true,"origin":"","legend":"Verification of the SmAP2/ERF genes with differentially expressed patterns under salt stress by quantitative real-time PCR \nFor salt stress, ‘Yanjiang’ and ‘9901’ roots that were subjected to 20 days of hydroponic culture and then treated with 150 mM sodium chloride for 4 h. The control was an untreated Yanjiang sample. Three biological replicates for each sample were performed, and bars represent the standard deviations of the mean. Asterisks on top of the bars indicate statistically significant differences between stress treatment and the control (*0.01 \u003c P \u003c 0.05; **p\u003c0.01, Student’s t-test). Gene expression profiles were evaluated using the 2−∆∆Ct method, and the control value was normalized to 1.","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/0f5e8f94-007b-4a68-8ffc-04ca646c5fa4/v1/fig8.png"},{"id":15666072,"identity":"9469ac49-5201-43b1-8fe3-5991275d90c2","added_by":"auto","created_at":"2021-11-18 13:35:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3922578,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-15225/v1/1359c215-e4b1-4a1d-b887-410ec4c81eda.pdf"},{"id":554833,"identity":"df6f3b72-d05b-4e81-836e-b2f568d3d50e","added_by":"auto","created_at":"2020-02-27 02:21:26","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1524584,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalfilesTableS1S7FigureS1S2.docx","url":"https://assets-eu.researchsquare.com/files/0f5e8f94-007b-4a68-8ffc-04ca646c5fa4/v1/Supplemental files Table S1-S7 Figure S1 S2.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eGenome-wide investigation of the AP2/ERF superfamily and their expression under salt stress in Chinese willow (\u003cem\u003eSalix matsudana\u003c/em\u003e)\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAP\u003c/span\u003eETALA 2/\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ee\u003c/span\u003ethylene-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003er\u003c/span\u003eesponsive element binding \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ef\u003c/span\u003eactors (AP2/ERF) are important transcription factors (TFs) coded by genes from the AP2/ERF superfamily. All of the members of this superfamily possess AP2 domains and, according to the number and structure of AP2 domains, the superfamily is divided into several categories, including AP2, ERF, RAV, and Soloist [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Most of the AP2 gene family members have two AP2 domains and can be further divided into AP2 and ANT groups; ERF family members have only one AP2 domain and can also be subdivided into ERF and DREB subfamilies based on binding motifs in the promoter of downstream genes. Members of the ERF and DREB subfamilies are classified into 12 groups (groups A1\u0026ndash;B6). DREB includes groups A1\u0026ndash;A6, whereas ERF includes groups B1\u0026ndash;B6 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In addition to one AP2 domain, RAVs also have one B3 domain. The Soloist group contains a single AP2 domain with sequence divergence from the AP2 and ERF families and has less than three members in most species [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe AP2/ERF superfamily is plant-specific and has more than 100 members in many plant species; for example, there are 147 members in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eArabidopsis\u003c/span\u003e, 200 members in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e, and more than 500 members in the tetraploid crop \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eBrassica napus\u003c/span\u003e [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Different members play various regulatory roles in plant growth and development, defense response, fruit ripening, and metabolism [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Several recent reports demonstrated functions of AP2/ERF2 TFs in plant development. For example, loss of DRNL function affects gynoecium development [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]; the function of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus\u003c/span\u003e ERF139 (Potri.013G101100) in xylem cell expansion was characterized by transgenic overexpression and dominant repressor lines of ERF139 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]; RhERF1 and RhERF4 play roles in petal abscission in rose [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]; and a maize AP2/ERF TF, ZmRAP2.7, is involved in brace root development. AP2/ERF TFs such as ZmEREB94 and CitAP2.10 also play important roles in plant metabolism; ZmEREB94 acts as a key regulator of starch synthesis in maize [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and CitAP2.10 was characterized as a regulator of (+)-valencene synthesis in sweet orange fruit [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe AP2/ERF superfamily plays major and crucial roles in abiotic stress tolerance, which is why this superfamily has received special attention by plant scientists [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Through extensive investigation on their regulatory mechanism, people want to elucidate their potential applications in crop improvement [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Members of this superfamily (primarily ERFs and DREBs) have been prominently used to improve stress tolerance in plants. To improve salinity stress tolerance, many genes from different species were identified. IbRAP2-12, an AP2/ERF gene cloned from the salt-tolerant sweet potato, and LkERF-B2 from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eLarix kaempferi\u003c/span\u003e promote tolerance to salt and drought stresses in overexpressing \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eArabidopsis\u003c/span\u003e lines [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Overexpression of HARDY, an AP2/ERF gene from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eArabidopsis\u003c/span\u003e, improves drought and salt tolerance by reducing transpiration and sodium uptake in transgenic \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eTrifolium alexandrinum L\u003c/span\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. A soybean DREB ortholog, GmDREB1, enhances the salt tolerance in transgenic alfalfa [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eComparative genomic analysis of model plants such as \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eArabidopsis\u003c/span\u003e have provided unprecedented advantages for gene discovery and functional annotation of newly sequenced plant genomes [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. By exploring the available genomic data, AP2/ERF gene families from 50 species were discovered and classified, and provide critical guidance for functional analysis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. For example, in radish, cauliflower, and celery, whole genome identification and classification of AP2/ERF gene family members were carried out; additionally, expression patterns of different members under different stresses were revealed, and the function of candidate genes was verified [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana Koidz\u003c/span\u003e., a member of Salicaceae, is an important ornamental tree species native to northeastern China [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]; it is widely cultivated and considered an important economic plant because of its easy vegetative propagation, rapid growth, and substantial biomass yields. \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e also plays an important ecological role when grown along Chinese coastal beaches, where the salinity content is high [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This species can improve the beach soil and alleviate salinization. Newly reclaimed beach soil has higher salinity and requires new germplasm with higher salinity tolerance [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Because the AP2/ERF gene family members have regulatory roles in salinity tolerance, whole genome characterization of the AP2/ERF gene family in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e will reveal mechanisms underlying stress signal transmission and provide guidance for selection or creation of new germplasm with higher salinity tolerance. In total, 200 and 173 AP2/ERF superfamily genes were identified from two species, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix arbutifolia\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e, respectively [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e genome was recently sequenced and assembled (unpublished); as a tetraploid, identification of the AP2/ERF gene family will reveal the evolutionary relationship with poplar and other members of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix\u003c/span\u003e, and the molecular mechanisms responsible for salinity stress responses.\u003c/p\u003e "},{"header":"Results","content":" \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eIdentification, phylogenetic analysis, and classification of 364 AP2/ERF TF family memebers in\u003c/span\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e\u003c/p\u003e \u003cp\u003eBy HMM profile search against the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e protein database, a total of 364 full-length AP2/ERF family proteins containing at least one AP2/ERF domain were identified as AP2/ERF superfamily members of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The name, protein length, molecular weight, and isoelectric point of individual genes are listed in Supplementary Table S1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe phylogenetic relationships of SmAP2/ERF proteins were inferred by multiple sequence alignment of the AP2 domain, which included approximately 50\u0026ndash;60 amino acids. The sequence alignment of all AP2/ERF genes showed some conserved amino acids at specific positions, as previously reported (Fig. S1). For example, the WLG element (58th\u0026ndash;60th amino acids; 58\u0026ndash;60AA) was highly conserved in in the ERF and RAV families; alternatively, in the AP2 family, the conserved sequences from 58\u0026ndash;60AA were converted into YLG elements [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In many species, these conserved amino acid profiles contribute to convincing classification of AP2/ERF genes. Basing on multiple sequence alignments of 48 AP2/ERF proteins from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eArabidopsis\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e with known categories and 364 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e AP2/ERF proteins, we constructed a phylogenetic tree using the neighbor-joining method to explore the phylogenetic relationships of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e AP2/ERF proteins. The phylogenetic tree showed that there were 55 AP2/ERF genes that belong to the AP2 family, with 47 genes that encode proteins with two AP2 domains and eight genes (SmAP2-20, SmAP2-25, SmAP2-29, SmAP2-35, SmAP2-36, SmAP2-40, SmAP2-41 and SmAP2-55) that encode proteins with a single AP2 domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Additionally, 301 genes that were predicted to encode proteins with a single AP2 domain were members of the ERF family. The ERF family could be further classified into two subfamilies, ERF and DREB. Of the 301 members, 166 and 135 genes belonged to the ERF and DREB subfamilies, respectively. The ERF family genes from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e were distributed in B1\u0026ndash;B6 subgroups; the DREB family genes from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e were classified into A1\u0026ndash;A6 subgroups. The gene number and percentage of each subgroup are listed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table S2. Six putative genes were classified as RAV subgroup genes that encode proteins containing one AP2/ERF domain and one B3 domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Two genes were designated as Soloist genes, whose AP2/ERF-like domain sequences had lower homology compared with other AP2/ERF genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe AP2/ERF genes number, classification and percentage of different subgroups from five plant species, including the model plant \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eArabidopsis\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus\u003c/span\u003e, and two \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix\u003c/span\u003e plants, are listed in Table S2. The gene name of AP2/ERF genes from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e are listed in Table S3. As a tetraploid plant, the total number (364) of AP2/ERF genes was much larger in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e than in the other four species. The number of AP2/ERF genes in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e was 2.5-, 1.8-, 1.9-, and 2.1-fold higher than those in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eA. thaliana\u003c/span\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix arbutifolia\u003c/span\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], respectively. For DREB and ERF subfamilies, the percentage of all AP2/ERF genes in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e was similar to those of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eA. thaliana\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e, and the percentages of DREB and ERF subfamilies were 38% and 45%, respectively. In \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix arbutifolia\u003c/span\u003e, the percentage of DREB (33%) was lower than that of the other four species, whereas the percentage of ERF (50.8%) was higher. In \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e, the percentage of the AP2 subgroup was highest among all five species (15%) and the numbers of most of gene sub-classifications were doubled, including the Soloist gene; there were two Soloist genes in the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e genome. However, no duplications were observed in the RAV subgroup, and only six RAV genes were found in the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e genome.\u003c/p\u003e \n\u003cP\u003eGene Structure And Conserved Motif Analysis\u003c/p\u003e\n \u003cp\u003eTo understand the structural diversity of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2/ERF\u003c/span\u003e genes in different clades, the intron and exon structures of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2/ERF\u003c/span\u003e genes were revealed by inputting Gff3 files into TBtools (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). A total of 55 genes of the AP2 subfamily had more exons than ERF and other subfamilies. Apart from three exons in the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2-29\u003c/span\u003e and four exons in the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2-20\u003c/span\u003e, other members of the AP2 subfamily contained more than seven exons. The intron number was less than three in many members of the ERF and RAV subfamilies. In total, 215 gene members did not have introns (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The exon/intron structures of genes that were classified in the same clade were similar. Many gene pairs were found in the phylogenetic tree that potentially originated from allotetraploid or autotetraploid evolution of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e. Many gene pairs (approximately 70%) maintained the same or similar gene structure during \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e formation, which indicated that the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2/ERF\u003c/span\u003e genes were conserved at the DNA level during polyploidization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTF proteins always contain many conserved motifs to activate gene expression. A total of 10 conserved motifs were detected in 364 SmAP2/ERF proteins using the online MEME software, and a block diagram was constructed to characterize SmAP2/ERF protein structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Motif-4, Motif-1, Motif-2, Motif-3, Motif-5, Motif-7, and Motif-9 were found in the AP2 domain regions. The Motif-5 region covered the region of Motif-4 and Motif-1, whereas Motif-7 included Motif2 and Motif3. Motif-9 is a specific motif that is only found in the second AP2 domain of the AP2 subgroup. Motif-1, Motif-2, Motif-3, and Motif-4 were detected in 90% percent of the ERF subfamily proteins. Thirty proteins of the ERF subfamily lacked one or two motifs of Motif-1\u0026ndash;4. An extreme example is \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmERF B2-13\u003c/span\u003e, which only had Motif-2. Motif-6, Motif-8, and Motif-10 are motifs located outside of the AP2 domain. Motif-6 was primarily found in the AP2 subfamily with only one exception, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmERF B4-4\u003c/span\u003e, which was in the ERF-B4 clade. In the AP2 subfamily, members with two AP2 domains had Motif-6 located between the two AP2 domains. Motif-8 was found in 69 proteins of the AP2/ERF family, and its location was adjacent to the carboxyl terminal of Motif-3. Many proteins from the DREB-A1, DREB-A4, DREB-A5 clades had Motif-8. Motif-10 was found in 62 proteins of the AP2/ERF family, with 61 proteins from the ERF subfamily and only one from the AP2 subfamily. Motif-10 was mostly distributed on the proteins from the ERF-B3, DREB-A2, and DREB-A4 clades. The functions of these three motifs need to be elucidated by further experimental analysis.\u003c/p\u003e \u003cp\u003eBesides protein SmAP2-20, the entire AP2 domain was distributed in the amino terminal or in the middle position of the proteins. In the two Soloist genes, only one motif, Motif-2, was found.\u003c/p\u003e \u003cp\u003eThe conserved motif composition and gene structure of the same subfamily were similar, thus verifying the reliability of the phylogenetic tree classification.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eChromosome distribution and duplication of\u003c/span\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eSmAP2/ERF\u003c/span\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003esuperfamily genes\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe chromosome location of the identified \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2/ERF\u003c/span\u003e genes was constructed using TBtools. In total, 310 genes from the AP2/ERF superfamily were unevenly distributed on 38 chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e); 54 other genes located on scaffolds were not illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The chromosome with the largest number of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eAP2/ERF\u003c/span\u003e genes was Chr21, which had 22 genes. Only one \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eAP2/ERF\u003c/span\u003e gene each was located on Chr14 and Chr36. On the four chromosomes Chr1, Chr3, Chr22, and Chr27, only two \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eAP2/ERF\u003c/span\u003e genes were found. In 38 chromosomes, most of the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eAP2/ERF\u003c/span\u003e genes from different subgroups were arbitrarily distributed, such as five of six RAV genes located on Chr15, Chr37, Chr34, Chr31, and Chr11. Moreover, the two Soloist genes distributed on Chr29 and Chr5. However, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmERF\u003c/span\u003e B3 subgroup members clustered together with three genes as a cluster unit. We found 12 clusters in 12 chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), which accounted for 62% of the whole \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmERF B3\u003c/span\u003e subgroup.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, we also analyzed the tandem duplication events (TDs) of the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eAP2/ERF\u003c/span\u003e genes located within in the 200-kb range of chromosomal regions of the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e genome. Eleven TD regions, which included 23 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2/ERF\u003c/span\u003e genes, clustered into 11 linkage groups (LGs) of the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). LGs that contained cluster repeat genes were Chr7, Chr8, Chr10, Chr13, Chr17, Chr19, Chr21, Chr24, Chr28, Chr31, and Chr33. All genes of the repeat clusters were localized within a genomic segment of less than 20 Kb; for example, SmDREB A4-20 and SmDREB A4-19 were localized on a 3.6-Kb segment of Chr24. On Chr8, three genes clusters (SmERF B3-6, SmERF B3-7, and SmERF B3-8) located on a less than 12-Kb segment. In 11 tandem repeats, six came from the SmERF B3 subgroup, two came from the SmDREB A4 subgroup, and one each came from the SmDREB A1 and AP2 subgroups. SmERF B3-40 and SmERF B3-39 tandem repeat pairs had 97% protein sequence identity.\u003c/p\u003e \u003cp\u003eIn addition to tandem duplications, many segmental duplication events (SDs) were found in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e by MCScanX (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table S4). We found a total of 28,348 collinear gene pairs (not shown) in the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e genome, from which 298 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eAP2/ERF\u003c/span\u003e collinear gene pairs were identified. Then, Ka, Ks, and Ka/Ks ratios of these 298 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eAP2/ERF\u003c/span\u003e collinear gene pairs were calculated to estimate the divergence time (T value) and selection pressure among duplicated \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2/ERF\u003c/span\u003e gene pairs (Table S5). All of the Ka/Ks values were below 1, which indicated that these genes might have experienced strong purifying selective pressure during evolution. Among the 298 AP2/ERF collinear gene pairs, 198 were located on duplicated segments on 38 chromosomes in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Table S4). The collinear gene pairs in the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e genome were visualized by Circos, and the gene pairs were linked by lines (grey lines indicated all gene pairs, red lines indicated \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eAP2/ERF\u003c/span\u003e collinear gene pairs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eSynteny analysis of\u003c/span\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eAP2/ERF\u003c/span\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003egenes between\u003c/span\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eand two related Salicaceae species\u003c/span\u003e, \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eand\u003c/span\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e\u003c/p\u003e \u003cp\u003eTo further infer the phylogenetic mechanisms of the SmAP2/ERF family, we constructed two comparative syntenic maps of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e with two related species, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).Collinear \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eAP2/ERF\u003c/span\u003e genes pairs between \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e and other two species are listed in Supplementary Table S6. A total of 263 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2/ERF\u003c/span\u003e genes showed syntenic relationships with 183 genes from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e, and 248 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2/ERF\u003c/span\u003e genes showed syntenic relationships with 144 genes from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e. The number of orthologous pairs between \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e were 423 and 292, respectively (Supplementary Table S6). Some \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePtAP2/ERF\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSpAP2/ERF\u003c/span\u003e genes were found to be associated with at least four syntenic gene pairs. Interestingly, the number of collinear gene pairs identified between \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e were less than that between \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the comparative syntenic map between \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e, syntenic links were found between all 19 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e chromosomes and all 38 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e chromosomes. Alternatively, in the comparative syntenic map between \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e, there were no syntenic links between Chr1, Chr12, and Chr36 from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e, and Chr15Z and Chr15W from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \n\u003cp\u003eSpecific Expression Of AP2/ERF Superfamily Genes Under Salt Stress\u003c/p\u003e\n \u003cp\u003eTo investigate the physiological roles of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2/ERF\u003c/span\u003e genes in salt stress tolerance, we identified the expression patterns of SmAP2, SmERF, and SmDREB subgroup genes from the RNA sequencing data. By inputting the FPKM values (Fragments Per Kilobase of transcript per Million fragments mapped) of these genes in TBtools, three heatmaps were constructed to demonstrate the expression pattern change under salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression patterns of 286 genes are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, and included 48 AP2, 108 DREB, and 130 ERF subgroup genes. In the AP2 subgroup, the FPKM values of 31 genes were \u0026lt;\u0026thinsp;3, which indicated lower expression in the root and no response to salt stress. Five genes had differential expression patterns. The expression levels of four genes (\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2-38\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2-4\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2-3\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2-33\u003c/span\u003e) were induced by salt stress, whereas the expression of gene \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2-15\u003c/span\u003e decreased after salt stress. In the DREB subgroup, 108 genes were present in the heatmap, and expression levels of 10 genes, such as \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmDREB A1-10\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmDREB A1-9\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmDREB A1-7\u003c/span\u003e, were induced by salt stress and remained higher. In the ERF subgroup heatmap, 130 genes were included. The expression levels of 13 genes were upregulated by salt stress, but only the expression of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmERF B4-1\u003c/span\u003e was higher. Three genes were downregulated by salt stress, including \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmERF B3-52\u003c/span\u003e. In many paralog gene pairs, we found one gene with higher expression, whereas the other gene had lower expression, such as \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmDREB-A9\u003c/span\u003e/ \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmDREB-A10\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2-33\u003c/span\u003e/\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2-39\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmERF-9\u003c/span\u003e/ \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmERF-10\u003c/span\u003e gene pairs. Thirteen Genes with upregulated expression patterns was verified by qRT-PCR (Real-time Quantitative PCR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003e \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix\u003c/span\u003e is one of the few woody plants with a large number of polyploid taxa, in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e, both tetraploid and diploid individuals have been observed. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In our experiment, we sequenced the genome of tetraploid \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e. Tetraploid \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix\u003c/span\u003e have value because they have higher tolerance to abiotic stress than their diploid relatives; therefore, they can be planted beachside to alleviate soil salinity and improve the ecological environment [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The molecular mechanism of salinity response regulation is very complex, and AP2/ERF TFs are key regulators in plants [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Here, we identified 364 AP2/ERF gene members in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e, and characterized their classification, chromosome location, gene structure, and syntenic relationships of these genes within the genome and between other species. We also revealed the expression patterns under salt stress. These efforts can serve as a first step in comprehensive functional characterization of AP2/ERF genes by reverse genetic approaches and molecular genetics research.\u003c/p\u003e \u003cp\u003eAs a tetraploid species, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e has more AP2/ERF gene members than other plants selected for comparison, including three Salicaceae family relatives (Table S2). The total number of genes is approximately double compared with poplar and two willow relatives, but the proportions of some subgroups were slightly different. \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix arbutifolia\u003c/span\u003e had a higher percentage (50.8% \u0026gt; 45%) of ERF subfamily members, but a lower proportion (33% \u0026lt; 38%) of DREB subfamily members compared with other species (Table S2). For DREB-A1 and ERF-B2 subgroups, the highest percentage was found in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e, and there were the same number of or more members of these two subgroups compared with other species, including the tetraploid \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e. For ERF-B3, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix arbutifolia\u003c/span\u003e had the highest subgroup percentage (18.5%). In \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e, ERF-B6 had the lowest percentage (5.7%), whereas the subfamily AP2 had the highest percentage (15.1%). These data indicated that, during evolution, AP2/ERF family subgroup members probably underwent gene duplication or loss and therefore evolved into the specific AP2/ERF subgroup proportions in each species.\u003c/p\u003e \u003cp\u003eA phylogenetic tree that included 364 genes from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e and 48 genes from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eA. thaliana\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e was constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All subgroups were clustered together. Eight genes with one AP2 domain were classified into the AP2 family because of a close phylogenetic relationship. This classification was similar to that in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eArabidopsis\u003c/span\u003e, in which four genes involved in the AP2 family contained a single AP2 domain [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe gene intron/exon structure and conserved motifs were identified in the 364 SmAP2/ERF members. Similar to that of the AP2/ERF genes from other species, such as cauliflower and radish, the AP2 subfamily had more introns and the ERF subfamily had fewer [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Previous studies found that intron number and distribution are related to plant evolution, and introns of the ERF family genes were probably lost during evolution in higher plants [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In total, 215 of the 301 members (70%) of the ERF family had no introns, which was a little less than that of tartary buckwheat [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and also consistent with previous findings.\u003c/p\u003e \u003cp\u003eThrough the conserved domains and motifs, TFs play roles in gene expression regulation by promoter binding, transcription activation, and protein\u0026ndash;protein interactions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Motif analysis showed that Motif-6, Motif-8, and Motif-10 were specifically detected in different groups of the AP2/ERF subfamily; seven other motifs were all related to the AP2 domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Motif-8 was specifically found in the DREB subgroup, such as in the DREB-A1, DREB-A4, and DREB-A5 clades. Motif-10 was mostly found distributed on proteins from the ERF-B3, DREB-A2, and DREB-A4 clades. Motif-6 was specifically located between the two AP2 domains of AP2 subgroup members. These results indicate that, although some motifs of the AP2/ERF family genes were highly conserved and involved in DNA binding, such as motifs from the AP2 domain, the functions of other subgroup-specific motifs are still unknown, and more work is required to clarify their regulatory functions.\u003c/p\u003e \u003cp\u003eBased on the genome assembly data, 301 genes were anchored on the 38 chromosomes (LGs), but they were unevenly distributed. Eleven TDs were found on 11 chromosomes, and seven tandem duplication gene pairs came from the SmERF B3 subgroup, which included three duplicated genes (\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmERF B3-6\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmERF B3-7\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmERF B3-8\u003c/span\u003e) that clustered together. Apart from the tandem duplication cluster, SmERF B3 members typically clustered on a chromosome, with three genes as a unit. In 12 clusters, 37 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmERF B3\u003c/span\u003e genes were found. This phenomena were also found in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e, thirteen PtERF B3 genes located in 4 clusters, which indicated that in the evolution of Sm, apart from the chromosome duplication, segmental duplication were also happened.\u003c/p\u003e \u003cp\u003eUsing MCScanX, we found a total of 28,348 collinear gene pairs in the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e genome, from which 299 AP2/ERF collinear gene pairs were identified; this indicated that, during evolution, the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e genome experienced a whole genome duplication event. Population genetic theory predicts that, after duplication, some redundant duplicate copies will be silenced and eliminated, and other retained paralogs will obtain sub- or neofunction by DNA mutation in coding or regulatory sequences [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThen, we calculated Ka, Ks, and Ka/Ks ratios of these 298 AP2/ERF collinear gene pairs to estimate the divergence time and selection pressure. All Ka/Ks values were below 1, which indicated that these genes might have experienced strong purifying selective pressure during evolution. It was previously reported that purifying selection would lead to the loss of redundant genes [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Based on the gene number of most subgroups, we did not find any obvious evidence of gene loss, but in the RAV subgroup, there was an exception; there were only six members in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e, which is identical to the gene number in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eArabidopsis\u003c/span\u003e. Based on the gene loss hypothesis, the duplication paralogs of RAVs may have been lost during genome evolution because of their rapid evolutionary rate.\u003c/p\u003e \u003cp\u003eApproximately 52\u0026ndash;59\u0026nbsp;million years ago (Mya), willow and poplar, which are two modern taxa, originated from a diploid progenitor, but when and how \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e experienced chromosome duplication remains largely unknown [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The divergence time (T Value) of gene pairs mainly occurred during two time periods, 2\u0026ndash;8 and 20\u0026ndash;36 Mya. Gene pairs with a divergence time of 20\u0026ndash;36 Mya were probably paralogs before whole genome duplication events, whereas 2\u0026ndash;8 Mya is probably the divergence time of paralogs after whole genome duplication events. These data indicated that the progenitor of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e underwent whole genome duplication not more than 10 Mya.\u003c/p\u003e \u003cp\u003eSimilar to the findings of a previous report [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], alignment of a \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix\u003c/span\u003e linkage map to the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus\u003c/span\u003e genomic sequence revealed macrosynteny between willow and poplar genomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Synteny analysis of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e vs \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e vs \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e revealed 423 and 292 orthologous pairs, respectively. In total, 263 SmAP2/ERF genes had syntenic relationships with 183 genes in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e, whereas 248 SmAP2/ERF genes showed syntenic relationships with 144 genes in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e. Interestingly, the collinear gene pairs identified between \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e were less than that from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e. Syntenic links were found between all 19 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e chromosomes and all 38 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e chromosomes, but there were no syntenic links between Chr1, Chr12, and Chr36 from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e and Chr15Z and Chr15W from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e. \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix\u003c/span\u003e has 300\u0026ndash;500 species and considerable variation, ranging from shrubs to trees [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]; willow may evolve faster, which would lead to them being more diverse. Researchers proposed that \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus\u003c/span\u003e might be evolutionarily more primitive than \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix\u003c/span\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. From our results, we could infer the evolutionary relationships of three Salicaceae species (\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e); \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e was the most primitive taxon, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e was the most derived taxon, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e was located between them but was genetically more closely related to \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e than \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e.\u003c/p\u003e \u003cp\u003ePlants must adapt to various biotic and abiotic stresses because they are immobile in their life cycles. For example, \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e must adapt to the soil salinity when grown along coastal beaches. Consequently, some AP2/ERF TFs play important roles in plants by facilitating defense against stress and improving resistance. From the RNA sequencing data, we extracted the expression FPKM values and constructed expression heatmaps to show the expression patterns under salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The expression levels of four genes from the AP2 subgroup, 10 genes from the DREB subgroup, and 13 genes from the ERF subgroup were induced by salt stress, but only the expression levels of four genes were downregulated after salt stress. The expression patterns were verified by qRT-PCR. The expression pattern of many AP2/ERF gene pairs with evolutionary relationships differed, which indicated that the AP2/ERF gene family may have changed at the transcriptional regulation level followi polyploidization. That finding provides additional evidence that redundant duplicated gene pairs experienced functional divergence based on expression pattern change. These differentially expressed SmAP2/ERF genes could be selected as candidate genes; further exploration on their roles under salt stress will reveal molecular mechanisms responsible for salinity stress responses in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, 364 AP2/ERF TFs were identified in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e. Clustering and phylogenetic analysis were conducted to classify these TFs into 15 subgroups. Chromosome location, gene structure, and conserved motifs were identified for 364 AP2/ERF TFs. Evolutionary relationships of these genes were revealed by tandem and segmental duplication gene pair identification, divergence time estimation, and T value calculation, which indicated that the progenitor of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e underwent whole genome duplication not more than 10 Mya. Synteny analysis with other species showed macrosynteny between willow and poplar AP2/ERF genes, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e was genetically more closely related to \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e than \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e. The AP2/ERF TFs were also confirmed to exhibit differential expression patterns during salt stress. The functions of these genes should be investigated in future studies to better clarify the mechanism of salt tolerance regulation in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eIn this study, 364 \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2/ERF\u003c/span\u003e genes of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e were identified and renamed according to the chromosomal location of the SmAP2/ERF genes. Gene classification, gene structure and conserved motifs were analyzed in detail. Investigation results on syntenic relationships between the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2/ERF\u003c/span\u003e genes and AP2/ERF genes from other species elucidated that the progenitors of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e underwent whole genome duplication not more than 10 Mya and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e is genetically more closely related to \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e than to \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e. Moreover, analyses on the differential expression patterns of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSmAP2/ERF\u003c/span\u003e genes during salt stress can help to reveal the mechanism of salt tolerance regulation in \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e.\u003c/p\u003e "},{"header":"Methods","content":" \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cp\u003eMaterial and salt stress treatment\u003c/p\u003e \u003cp\u003eThe salinity stress treatments were carried out on \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e \u0026lsquo;yanjiang\u0026rsquo; and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e \u0026lsquo;9901\u0026rsquo;. The 2 salix cultivars used in this study were collected from the botany garden of Nantong University (Nantong, China). The 2 salix cultivars were authorized for only scientific research purpose, and were deposited in College of life science in Nantong University. The stem cuttings (length, 8\u0026ndash;10\u0026nbsp;cm; coarse, 2\u0026ndash;3\u0026nbsp;cm) of two materials were selected for hydroponic rooting for 20 days and then treated with NaCl solution (150\u0026nbsp;mM) for 4\u0026nbsp;h. All root samples were divided into four categories with three biological replicates: Sample1-1/Sample1-2/Sample1-3 (\u0026lsquo;yanjiang\u0026rsquo; without salt stress treatment), Sample1N-1/Sample1N-2/Sample1N-3 (\u0026lsquo;yanjiang\u0026rsquo; with salt stress treatment), Sample2-1/Sample2-2/Sample2-3 (\u0026lsquo;9901\u0026rsquo; without salt stress treatment), and Sample2N-1/Sample2N-2/Sample2N-3 (\u0026lsquo;9901\u0026rsquo; with salt stress treatment).\u003c/p\u003e \u003c/div\u003e \n\u003cp\u003eGenome Sequence Retrieval\u003c/p\u003e\n \u003cp\u003eThe \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e sequences were downloaded from JGI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.phytozome.net/\u003c/span\u003e\u003c/span\u003e). The \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e sequences were obtained from our sequencing, and assembly results were obtained by Roche/454 and Illumina/HiSeq-2000 sequencing technologies (unpublished).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eIdentification of AP2/ERF genes in\u003c/span\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eand\u003c/span\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe Pfam accession number of AP2 domain is PF00847.16. We downloaded the Hidden Markov Model (HMM) profile for the AP2/ERF TFs from the Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org/\u003c/span\u003e\u003c/span\u003e) with Pfam accession number PF00847.16 as the search keyword. An alternative HMM profile was built by sequence alignment using Clustal\u003c/p\u003e \u003cp\u003eW[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Using an in-house Perl script with two HMM profiles as queries, hmmsearch was carried out by searching the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e protein databases with default parameters. To validate the putative accuracy of two HMM search results, the candidate protein sequences were checked in three websites: SMART (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl.de/#\u003c/span\u003e\u003c/span\u003e), CDD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/cdd/\u003c/span\u003e\u003c/span\u003e), and Pfam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org/\u003c/span\u003e\u003c/span\u003e). Candidate proteins with positive results from all three websites were selected as AP2/ERF family members of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e. Additionally, putative AP2/ERF protein characteristics, including length, molecular weight, and isoelectric point, were calculated by the ExPasy site (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://au.expasy.org/tools/pi_tool.html\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \n\u003cp\u003ePhylogenetic Analysis And Classification Of AP2/ERF Genes\u003c/p\u003e\n \u003cp\u003eUsing an in-house Perl script (domain_xulie.pl), the conserved AP2 core domains of putative SmAP2 proteins were obtained and subjected to multiple sequence alignment using ClustalW [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. To better classify these SmAP2 genes, 48 AP2 domains from known categories of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eArabidopsis\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e AP2 genes were selected to carry out multiple sequence alignment with SmAP2 proteins, and a phylogenetic tree based on this alignment was built by MEGA 7.0 with the neighbor-joining method [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] with default parameters. Bootstrap value was set to 1000. Depending on the phylogenetic tree constructed by SmAP2, PtAP2, and AtAP2 domains, these SmAP2 genes were classified into different subfamilies and subgroups.\u003c/p\u003e \n\u003cp\u003eGene Structure And Conserved Motif Structure Analysis\u003c/p\u003e\n \u003cp\u003eThe UTR\u0026ndash;exon\u0026ndash;intron structures of the SmAP2 genes were obtained based on the gene annotation gff3 files we assembled. Using the online website tool Gene Structure Display Server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.cbi.pku.edu.cn/\u003c/span\u003e\u003c/span\u003e), we obtained the gene structure diagrams [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo characterize the SmAP2 protein structure, the online tool MEME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite.org/tools/meme\u003c/span\u003e\u003c/span\u003e) was used to search for conserved motifs [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The optimized parameters were employed as follows: any number of repetitions, maximum number of motifs\u0026thinsp;=\u0026thinsp;10, and the optimum width of each motif was 6\u0026ndash;50 residues. The search result file meme.xml was downloaded from the website and opened by TBtools v0.66831 to obtain the gene structure diagram [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eGene position on chromosomes, and gene tandem and segmental duplication analysis\u003c/span\u003e \u003c/p\u003e \u003cp\u003eUsing the \u0026ldquo;Amazing Gene Location From GFF3/GTF File\u0026rdquo; tool of TBtools, the SmAP2 genes were mapped on 38 chromosomes of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e. Because some scaffolds were not assembled onto the chromosomes, not all SmAP2 genes mapped onto the chromosomes.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e is a tetraploid willow. Gene duplication events are a common phenomenon in the genome. There are two kinds of gene duplications in the genome: tandem duplication events (TDs) and segmental duplication events (SDs). TDs refer to two or more adjacent homologous genes located within 200 Kb on a single chromosome; SDs refer to homologous gene pairs between different chromosomes [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The gene duplication pairs were identified in TBtools by the \u0026ldquo;Blast compare 2 Seq [sets]\u0026thinsp;\u0026lt;\u0026thinsp;Big File\u0026gt;\u0026rdquo; and \u0026ldquo;Quick McscanX Wrapper\u0026rdquo; tools. The candidate duplicated genes should have \u0026ge;\u0026thinsp;80% coverage and \u0026ge;\u0026thinsp;65% similarity. The TDs of SmAP2 genes were revealed on a chromosome using the \u0026ldquo;Amazing Gene Location From GFF3/GTF File\u0026rdquo; tool of TBtools. The SDs of SmAP2 genes were visualized by the \u0026ldquo;Amazing Super Circos\u0026rdquo; tool of TBtools.\u003c/p\u003e \n\u003cp\u003eDivergence Time Calculation Of Duplicated Genes\u003c/p\u003e\n \u003cp\u003eAfter BLASTn analysis of Coding sequences and obtaining duplicated gene pairs, the nonsynonymous (Ka) and synonymous (Ks) pairs were calculated by the \u0026ldquo;Simple Ka/Ks calculator (NG)\u0026rdquo; tool of TBtools. The divergence time was estimated with the formula: T\u0026thinsp;=\u0026thinsp;Ks/2λ. The clock-like rate λ value (9.1\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e) from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus\u003c/span\u003e was used in the calculation. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eCollinearity analysis between\u003c/span\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eand the representative species\u003c/span\u003e\u003c/p\u003e \u003cp\u003eTo demonstrate the syntenic relationships of the orthologous AP2 genes obtained from \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix matsudana\u003c/span\u003e and other two selected plants (\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ePopulus trichocarpa\u003c/span\u003e, and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea\u003c/span\u003e,), the syntenic analysis maps were constructed using the \u0026ldquo;Amazing Super Circos\u0026rdquo; tool of TBtools.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eRNA sequencing and a heat map generated by hierarchical clustering\u003c/span\u003e \u003c/p\u003e \u003cp\u003eTranscriptome sequencing data of 12 samples were obtained by Illumina HiSeq sequencing. The expression levels of genes in different samples were calculated using FPKM values. Using the \u0026ldquo;Amazing HeatMap\u0026rdquo; tool of TBtools, a graph of the expression level of AP2 family genes with hierarchical clustering was generated.\u003c/p\u003e \n\u003cp\u003eRNA Isolation And qRT-PCR Analysis\u003c/p\u003e\n \u003cp\u003eTotal RNA was extracted using TaKaRa MiniBEST Plant RNA Extraction Kit (Takara, Dalian, China) from roots according to the manufacturer\u0026rsquo;s instruction. For each sample, 3\u0026nbsp;\u0026micro;g of total RNA was used to synthesize first-strand cDNA with SuperScriptII reverse transcriptase (Takara, Dalian, China). For qRT-PCR, the reaction preparation, application parameter settings and quantitative analysis were performed as previously described [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The reactions were performed using the ABI Prism 7000 Real-time PCR system (Applied Biosystems, USA). The \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSalix purpurea Actin1\u003c/span\u003e gene (SapurV1A.0655s0050.1) were used as reference genes. The gene-specific primers for the 13 selected genes are listed in Table S7.\u003c/p\u003e "},{"header":"Abbreviations","content":" \u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAP2/ERF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAP2-like ethylene-responsive transcription factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFPKM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFragments Per Kilobase of transcript per Million fragments mapped\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eqRT-PCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReal-time Quantitative PCR\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMya\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emillion years ago\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHMM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHidden Markov Model\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTDs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etandem duplication events\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esegmental duplication events\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etranscription factors\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003cstrong\u003e.\u003c/strong\u003e\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\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eSalix matsudana\u003c/em\u003e genomic sequence datasets used during the current study are available from the corresponding author on reasonable request. Other data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (31971681), Jiangsu Province Forestry Science and Technology Innovation and Promotion Project (LYKJ [2018]36) and Nantong University Scientific Research Start-up project for Introducing Talents (18R08). The funding body has no role in the study design and data collection, analysis, interpretation of data and in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.C. and J.Z. conceived the research plans; Y.C. and Y.J. performed most of the experiments; G.L., C.Y., B.L., F.Z. and W.F. performed the rest of the experiments; Y.C. analyzed the data; Y.C. and J.Z. wrote the article. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Mallory Eckstut, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNakano T, Suzuki K, Fujimura T, Shinshi H: \u003cstrong\u003eGenome-wide analysis of the ERF gene family in Arabidopsis and rice\u003c/strong\u003e. \u003cem\u003ePlant Physiol \u003c/em\u003e2006, \u003cstrong\u003e140\u003c/strong\u003e(2):411-432.\u003c/li\u003e\n\u003cli\u003eSong X, Wang J, Ma X, Li Y, Lei T, Wang L, Ge W, Guo D, Wang Z, Li C\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eOrigination, Expansion, Evolutionary Trajectory, and Expression Bias of AP2/ERF Superfamily in Brassica napus\u003c/strong\u003e. \u003cem\u003eFront Plant Sci \u003c/em\u003e2016, \u003cstrong\u003e7\u003c/strong\u003e:1186.\u003c/li\u003e\n\u003cli\u003eZhuang J, Cai B, Peng RH, Zhu B, Jin XF, Xue Y, Gao F, Fu XY, Tian YS, Zhao W\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eGenome-wide analysis of the AP2/ERF gene family in Populus trichocarpa\u003c/strong\u003e. \u003cem\u003eBiochem Biophys Res Commun \u003c/em\u003e2008, \u003cstrong\u003e371\u003c/strong\u003e(3):468-474.\u003c/li\u003e\n\u003cli\u003eGu C, Guo ZH, Hao PP, Wang GM, Jin ZM, Zhang SL: \u003cstrong\u003eMultiple regulatory roles of AP2/ERF transcription factor in angiosperm\u003c/strong\u003e. \u003cem\u003eBotanical studies \u003c/em\u003e2017, \u003cstrong\u003e58\u003c/strong\u003e(1):6.\u003c/li\u003e\n\u003cli\u003eDuran-Medina Y, Serwatowska J, Reyes-Olalde JI, de Folter S, Marsch-Martinez N: \u003cstrong\u003eThe AP2/ERF Transcription Factor DRNL Modulates Gynoecium Development and Affects Its Response to Cytokinin\u003c/strong\u003e. \u003cem\u003eFront Plant Sci \u003c/em\u003e2017, \u003cstrong\u003e8\u003c/strong\u003e:1841.\u003c/li\u003e\n\u003cli\u003eWessels B, Seyfferth C, Escamez S, Vain T, Antos K, Vahala J, Delhomme N, Kangasjarvi J, Eder M, Felten J\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eAn AP2/ERF transcription factor ERF139 coordinates xylem cell expansion and secondary cell wall deposition\u003c/strong\u003e. \u003cem\u003eNew Phytol \u003c/em\u003e2019.\u003c/li\u003e\n\u003cli\u003eGao Y, Liu Y, Liang Y, Lu J, Jiang C, Fei Z, Jiang CZ, Ma C, Gao J: \u003cstrong\u003eRosa hybrida RhERF1 and RhERF4 mediate ethylene- and auxin-regulated petal abscission by influencing pectin degradation\u003c/strong\u003e. \u003cem\u003ePlant J \u003c/em\u003e2019, \u003cstrong\u003e99\u003c/strong\u003e(6):1159-1171.\u003c/li\u003e\n\u003cli\u003eLi H, Xiao Q, Zhang C, Du J, Li X, Huang H, Wei B, Li Y, Yu G, Liu H\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eIdentification and characterization of transcription factor ZmEREB94 involved in starch synthesis in maize\u003c/strong\u003e. \u003cem\u003eJ Plant Physiol \u003c/em\u003e2017, \u003cstrong\u003e216\u003c/strong\u003e:11-16.\u003c/li\u003e\n\u003cli\u003eShen SL, Yin XR, Zhang B, Xie XL, Jiang Q, Grierson D, Chen KS: \u003cstrong\u003eCitAP2.10 activation of the terpene synthase CsTPS1 is associated with the synthesis of (+)-valencene in 'Newhall' orange\u003c/strong\u003e. \u003cem\u003eJ Exp Bot \u003c/em\u003e2016, \u003cstrong\u003e67\u003c/strong\u003e(14):4105-4115.\u003c/li\u003e\n\u003cli\u003eSrivastava R, Kumar R: \u003cstrong\u003eThe expanding roles of APETALA2/Ethylene Responsive Factors and their potential applications in crop improvement\u003c/strong\u003e. \u003cem\u003eBriefings in functional genomics \u003c/em\u003e2018, \u003cstrong\u003e18\u003c/strong\u003e(4):240-254.\u003c/li\u003e\n\u003cli\u003eLi Y, Zhang H, Zhang Q, Liu Q, Zhai H, Zhao N, He S: \u003cstrong\u003eAn AP2/ERF gene, IbRAP2-12, from sweetpotato is involved in salt and drought tolerance in transgenic Arabidopsis\u003c/strong\u003e. \u003cem\u003ePlant Sci \u003c/em\u003e2019, \u003cstrong\u003e281\u003c/strong\u003e:19-30.\u003c/li\u003e\n\u003cli\u003eCao B, Shu L, Li A: \u003cstrong\u003eFunctional characterization of LkERF-B2 for improved salt tolerance ability in Arabidopsis thaliana\u003c/strong\u003e. \u003cem\u003e3 Biotech \u003c/em\u003e2019, \u003cstrong\u003e9\u003c/strong\u003e(7):263.\u003c/li\u003e\n\u003cli\u003eAbogadallah GM, Nada RM, Malinowski R, Quick P: \u003cstrong\u003eOverexpression of HARDY , an AP2/ERF gene from Arabidopsis, improves drought and salt tolerance by reducing transpiration and sodium uptake in transgenic Trifolium alexandrinum L\u003c/strong\u003e. \u003cem\u003ePlanta \u003c/em\u003e2011, \u003cstrong\u003e233\u003c/strong\u003e(6):1265-1276.\u003c/li\u003e\n\u003cli\u003eJin T, Chang Q, Li W, Yin D, Li Z, Wang D, Bao L, Liu L: \u003cstrong\u003eStress-inducible expression of GmDREB1 conferred salt tolerance in transgenic alfalfa\u003c/strong\u003e. \u003cem\u003ePlant Cell Tissue \u0026amp; Organ Culture \u003c/em\u003e2010, \u003cstrong\u003e100\u003c/strong\u003e(2):219-227.\u003c/li\u003e\n\u003cli\u003eBrendel V, Kurtz S, Walbot V: \u003cstrong\u003eComparative genomics of Arabidopsis and maize: prospects and limitations\u003c/strong\u003e. \u003cem\u003eGenome Biol \u003c/em\u003e2002, \u003cstrong\u003e3\u003c/strong\u003e(3):. reviews1005.1.\u003c/li\u003e\n\u003cli\u003eHall AE, Fiebig A, Preuss D: \u003cstrong\u003eBeyond the Arabidopsis genome: opportunities for comparative genomics\u003c/strong\u003e. \u003cem\u003ePlant Physiol \u003c/em\u003e2002, \u003cstrong\u003e129\u003c/strong\u003e(4):1439-1447.\u003c/li\u003e\n\u003cli\u003eSchranz ME, Song BH, Windsor AJ, Mitchell-Olds T: \u003cstrong\u003eComparative genomics in the Brassicaceae: a family-wide perspective\u003c/strong\u003e. \u003cem\u003eCurr Opin Plant Biol \u003c/em\u003e2007, \u003cstrong\u003e10\u003c/strong\u003e(2):168-175.\u003c/li\u003e\n\u003cli\u003eKaranja BK, Xu L, Wang Y, Tang M, M'Mbone Muleke E, Dong J, Liu L: \u003cstrong\u003eGenome-wide characterization of the AP2/ERF gene family in radish (Raphanus sativus L.): Unveiling evolution and patterns in response to abiotic stresses\u003c/strong\u003e. \u003cem\u003eGene \u003c/em\u003e2019, \u003cstrong\u003e718\u003c/strong\u003e:144048.\u003c/li\u003e\n\u003cli\u003eLi H, Wang Y, Wu M, Li L, Li C, Han Z, Yuan J, Chen C, Song W, Wang C: \u003cstrong\u003eGenome-Wide Identification of AP2/ERF Transcription Factors in Cauliflower and Expression Profiling of the ERF Family under Salt and Drought Stresses\u003c/strong\u003e. \u003cem\u003eFront Plant Sci \u003c/em\u003e2017, \u003cstrong\u003e8\u003c/strong\u003e:946.\u003c/li\u003e\n\u003cli\u003eLi MY, Liu JX, Hao JN, Feng K, Duan AQ, Yang QQ, Xu ZS, Xiong AS: \u003cstrong\u003eGenomic identification of AP2/ERF transcription factors and functional characterization of two cold resistance-related AP2/ERF genes in celery (Apium graveolens L.)\u003c/strong\u003e. \u003cem\u003ePlanta \u003c/em\u003e2019, \u003cstrong\u003e250\u003c/strong\u003e(4):1265-1280.\u003c/li\u003e\n\u003cli\u003eZhang J, Yuan H, Yang Q, Li M, Wang Y, Li Y, Ma X, Tan F, Wu R: \u003cstrong\u003eThe genetic architecture of growth traits in Salix matsudana under salt stress\u003c/strong\u003e. \u003cem\u003eHortic Res \u003c/em\u003e2017, \u003cstrong\u003e4\u003c/strong\u003e:17024.\u003c/li\u003e\n\u003cli\u003eZhang J, Yuan H, Li M, Li Y, Wang Y, Ma X, Zhang Y, Tan F, Wu R: \u003cstrong\u003eA High-Density Genetic Map of Tetraploid Salix matsudana Using Specific Length Amplified Fragment Sequencing (SLAF-seq)\u003c/strong\u003e. \u003cem\u003ePLoS One \u003c/em\u003e2016, \u003cstrong\u003e11\u003c/strong\u003e(6):e0157777.\u003c/li\u003e\n\u003cli\u003eRao G, Sui J, Zeng Y, He C, Zhang J: \u003cstrong\u003eGenome-wide analysis of the AP2/ERF gene family in Salix arbutifolia\u003c/strong\u003e. \u003cem\u003eFEBS open bio \u003c/em\u003e2015, \u003cstrong\u003e5\u003c/strong\u003e:132-137.\u003c/li\u003e\n\u003cli\u003eLiu M, Sun W, Ma Z, Zheng T, Huang L, Wu Q, Zhao G, Tang Z, Bu T, Li C\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eGenome-wide investigation of the AP2/ERF gene family in tartary buckwheat (Fagopyum Tataricum)\u003c/strong\u003e. \u003cem\u003eBMC Plant Biol \u003c/em\u003e2019, \u003cstrong\u003e19\u003c/strong\u003e(1):84.\u003c/li\u003e\n\u003cli\u003eGuo W, Hou J, Yin T, Chen Y: \u003cstrong\u003eAn analytical toolkit for polyploid willow discrimination\u003c/strong\u003e. \u003cem\u003eScientific reports \u003c/em\u003e2016, \u003cstrong\u003e6\u003c/strong\u003e:37702-37702.\u003c/li\u003e\n\u003cli\u003eXie Z, Nolan TM, Jiang H, Yin Y: \u003cstrong\u003eAP2/ERF Transcription Factor Regulatory Networks in Hormone and Abiotic Stress Responses in Arabidopsis\u003c/strong\u003e. \u003cem\u003eFront Plant Sci \u003c/em\u003e2019, \u003cstrong\u003e10\u003c/strong\u003e:228.\u003c/li\u003e\n\u003cli\u003eTang Y, Qin S, Guo Y, Chen Y, Wu P, Chen Y, Li M, Jiang H, Wu G: \u003cstrong\u003eGenome-Wide Analysis of the AP2/ERF Gene Family in Physic Nut and Overexpression of the JcERF011 Gene in Rice Increased Its Sensitivity to Salinity Stress\u003c/strong\u003e. \u003cem\u003ePlos One \u003c/em\u003e2016, \u003cstrong\u003e11\u003c/strong\u003e(3):e0150879.\u003c/li\u003e\n\u003cli\u003eZhang Z, Li X: \u003cstrong\u003eGenome-wide identification of AP2/ERF superfamily genes and their expression during fruit ripening of Chinese jujube\u003c/strong\u003e. \u003cem\u003eScientific reports \u003c/em\u003e2018, \u003cstrong\u003e8\u003c/strong\u003e(1):15612.\u003c/li\u003e\n\u003cli\u003eLiu L, ., White MJ, Macrae TH: \u003cstrong\u003eTranscription factors and their genes in higher plants functional domains, evolution and regulation\u003c/strong\u003e. \u003cem\u003eFebs Journal \u003c/em\u003e1999, \u003cstrong\u003e262\u003c/strong\u003e(2):247-257.\u003c/li\u003e\n\u003cli\u003eHou J, Wei S, Pan H, Zhuge Q, Yin T: \u003cstrong\u003eUneven selection pressure accelerating divergence of Populus and Salix\u003c/strong\u003e. \u003cem\u003eHorticulture Research \u003c/em\u003e2019, \u003cstrong\u003e6\u003c/strong\u003e(1):37.\u003c/li\u003e\n\u003cli\u003eXionglei H, Jianzhi Z: \u003cstrong\u003eRapid subfunctionalization accompanied by prolonged and substantial neofunctionalization in duplicate gene evolution\u003c/strong\u003e. \u003cem\u003eGenetics \u003c/em\u003e2005, \u003cstrong\u003e169\u003c/strong\u003e(2):1157-1164.\u003c/li\u003e\n\u003cli\u003eAdams KL, Richard C, Ryan P, Wendel JF: \u003cstrong\u003eGenes duplicated by polyploidy show unequal contributions to the transcriptome and organ-specific reciprocal silencing\u003c/strong\u003e. \u003cem\u003eP Natl Acad Sci USA \u003c/em\u003e2003, \u003cstrong\u003e100\u003c/strong\u003e(8):4649-4654.\u003c/li\u003e\n\u003cli\u003eKondrashov FA, Rogozin IB, Wolf YI, Koonin EV: \u003cstrong\u003eSelection in the evolution of gene duplications\u003c/strong\u003e. \u003cem\u003eGenome Biology \u003c/em\u003e2002, \u003cstrong\u003e3\u003c/strong\u003e(2):1-9.\u003c/li\u003e\n\u003cli\u003eHou J, Ye N, Dong Z, Lu M, Li L, Yin T: \u003cstrong\u003eMajor Chromosomal Rearrangements Distinguish Willow and Poplar After the Ancestral \u0026ldquo;Salicoid\u0026rdquo; Genome Duplication\u003c/strong\u003e. \u003cem\u003eGenome Biology \u0026amp; Evolution \u003c/em\u003e2016, \u003cstrong\u003e8\u003c/strong\u003e(6):1868-1875.\u003c/li\u003e\n\u003cli\u003eHanley SJ, Mallott MD, Karp A: \u003cstrong\u003eAlignment of a Salix linkage map to the Populus genomic sequence reveals macrosynteny between willow and poplar genomes\u003c/strong\u003e. \u003cem\u003eTree Genetics \u0026amp; Genomes \u003c/em\u003e2006, \u003cstrong\u003e3\u003c/strong\u003e(1):35-48.\u003c/li\u003e\n\u003cli\u003eArgus GW: \u003cstrong\u003eInfrageneric Classification of Salix (Salicaceae) in the New World\u003c/strong\u003e. \u003cem\u003eSystematic Botany Monographs \u003c/em\u003e1997, \u003cstrong\u003e52\u003c/strong\u003e:1-121.\u003c/li\u003e\n\u003cli\u003eDai X, Hu Q, Cai Q, Kai F, Ning Y, Tuskan GA, Milne R, Chen Y, Wan Z, Wang Z: \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\u003eLarkin M, Blackshields G, Brown N, Chenna R, Mcgettigan P, Mcwilliam H, Valentin F, Wallace I, Wilm A, R: \u003cstrong\u003eClustal W and clustal X version 2.0\u003c/strong\u003e. \u003cem\u003eBioinformatics \u003c/em\u003e2007, \u003cstrong\u003e23\u003c/strong\u003e(21):2947-2948.\u003c/li\u003e\n\u003cli\u003eKumar S, Stecher G, Tamura K: \u003cstrong\u003eMEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets\u003c/strong\u003e. \u003cem\u003eMolecular Biology \u0026amp; Evolution \u003c/em\u003e2016, \u003cstrong\u003e33\u003c/strong\u003e(7):1870.\u003c/li\u003e\n\u003cli\u003eHu B, Jin J, Guo AY, Zhang H, Luo J, Gao G: \u003cstrong\u003eGSDS 2.0: an upgraded gene feature visualization server\u003c/strong\u003e. \u003cem\u003eBioinformatics \u003c/em\u003e2014, \u003cstrong\u003e31\u003c/strong\u003e(8):1296.\u003c/li\u003e\n\u003cli\u003eBailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS: \u003cstrong\u003eMEME SUITE: tools for motif discovery and searching\u003c/strong\u003e. \u003cem\u003eNucleic Acids Res \u003c/em\u003e2009, \u003cstrong\u003e37\u003c/strong\u003e(Web Server issue):W202-208.\u003c/li\u003e\n\u003cli\u003eChen C, Xia R, Chen H, He Y: \u003cstrong\u003eTBtools, a Toolkit for Biologists integrating various HTS-data handling tools with a user-friendly interface\u003c/strong\u003e. \u003cem\u003ebioRxiv \u003c/em\u003e2018:doi: 10.1101/289660.\u003c/li\u003e\n\u003cli\u003eCannon SB, Mitra A, Baumgarten A, Young ND, May G: \u003cstrong\u003eThe roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana\u003c/strong\u003e. \u003cem\u003eBMC Plant Biology \u003c/em\u003e2004, \u003cstrong\u003e4\u003c/strong\u003e(1):10.\u003c/li\u003e\n\u003cli\u003eLynch M, Conery JS: \u003cstrong\u003eThe evolutionary fate and consequences of duplicate genes\u003c/strong\u003e. \u003cem\u003eScience \u003c/em\u003e2000, \u003cstrong\u003e290\u003c/strong\u003e(5494):1151-1155.\u003c/li\u003e\n\u003cli\u003eChen YH, Cao YY, Wang LJ, Li LM, Yang J, Zou MX: \u003cstrong\u003eIdentification of MYB transcription factor genes and their expression during abiotic stresses in maize\u003c/strong\u003e. \u003cem\u003eBiol Plantarum \u003c/em\u003e2018, \u003cstrong\u003e62\u003c/strong\u003e(2):222-230.\u003c/li\u003e\n\u003c/ol\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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Salix matsudana, SmAP2/ERF, Genome-wide, Salt tolerance, Expression patterns","lastPublishedDoi":"10.21203/rs.2.24667/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.24667/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e AP2/ERF transcription factors (TFs) play indispensable roles in plant growth, development, and especially in various abiotic stresses responses. The AP2/ERF TF family has been discovered and classified in more than 50 species, including model plants, horticulture plants, crops, and trees. Many AP2/ERF TFs from various species have been functionally characterized. However, little is known about the AP2/ERF gene family of Chinese willow (\u003cem\u003eSalix matsudana\u003c/em\u003e), which is a tetraploid ornamental tree species that is widely planted and is also considered a species that can improve the soil salinity of coastal beaches. The recently produced whole genome sequencing data of \u003cem\u003eSalix matsudana\u003c/em\u003e allowed us to conduct an evolutionary analysis on the AP2/ERF genes during polyploidization and study the genome-wide expression profiles of AP2/ERF genes in \u003cem\u003eSalix matsudana\u003c/em\u003e under salt stress. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e In this study, 364 AP2/ERF genes of \u003cem\u003eSalix matsudana\u003c/em\u003e (SmAP2/ERF) were identified and renamed according to the chromosomal location of the SmAP2/ERF genes. After phylogenetic analysis with known categories of genes from other species, the AP2/ERF genes were divided into three subfamilies: AP2 (55 members), ERF (301 members), and RAV (six members). Two Soloist genes were also identified. Gene structure and conserved motifs were analyzed in AP2/ERF genes, and introns were not found in most genes of the ERF subfamily, although some unique motifs were found to be important for the function of AP2/ERF genes. Syntenic relationships between the SmAP2/ERF genes and AP2/ERF genes from other species were also investigated to elucidate their evolutionary relationships during polyploidization. Moreover, analyses on the expression profiles under salt stress were also conducted.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThe progenitors of \u003cem\u003eSalix matsudana \u003c/em\u003eunderwent whole genome duplication not more than 10 Mya. Synteny analysis with other species showed macrosynteny between willow and poplar AP2/ERF genes and that \u003cem\u003eSalix matsudana \u003c/em\u003eis genetically more closely related to \u003cem\u003ePopulus trichocarpa\u003c/em\u003e than to \u003cem\u003eSalix purpurea\u003c/em\u003e. Further investigation of the AP2/ERF TFs demonstrated that differential expression patterns during salt stress and this information can help reveal the mechanism of salt tolerance regulation in \u003cem\u003eSalix matsudana\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Genome-wide investigation of the AP2/ERF superfamily and their expression under salt stress in Chinese willow (Salix matsudana)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-02-27 02:21:25","doi":"10.21203/rs.2.24667/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0d702a85-cdc9-4275-b8de-88771b57ce82","owner":[],"postedDate":"February 27th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":63041,"name":"Epigenetics \u0026 Genomics"}],"tags":[],"updatedAt":"2021-07-27T21:05:49+00:00","versionOfRecord":{"articleIdentity":"rs-15225","link":"https://doi.org/10.7717/peerj.11076","journal":{"identity":"peerj","isVorOnly":true,"title":"PeerJ"},"publishedOn":"2021-04-13 21:05:49","publishedOnDateReadable":"April 13th, 2021"},"versionCreatedAt":"2020-02-27 02:21:25","video":"","vorDoi":"10.7717/peerj.11076","vorDoiUrl":"https://doi.org/10.7717/peerj.11076","workflowStages":[]},"version":"v1","identity":"rs-15225","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-15225","version":["v1"]},"buildId":"J0_U0BvcaRcwD8yVFaRlm","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}