Genome-Wide Identification of the ERF Transcription Factor Family for Structure Analysis, Expression Pattern, and Response to Drought Stress in Populus alba × Populus glandulosa

The Ethylene Responsive Factor (ERF) transcription factor family is important for regulating plant growth and stress responses. Although the expression patterns of ERF family members have been reported in many plant species, their role in Populus alba × Populus glandulosa, an important model plant for forest research, remains unclear. In this study, we identified 209 PagERF transcription factors by analyzing the P. alba × P. glandulosa genome. We analyzed their amino acid sequences, molecular weight, theoretical pI (Isoelectric point), instability index, aliphatic index, grand average of hydropathicity, and subcellular localization. Most PagERFs were predicted to localize in the nucleus, with only a few PagERFs localized in the cytoplasm and nucleus. Phylogenetic analysis divided the PagERF proteins into ten groups, Class I to X, with those belonging to the same group containing similar motifs. Cis-acting elements associated with plant hormones, abiotic stress responses, and MYB binding sites were analyzed in the promoters of PagERF genes. We used transcriptome data to analyze the expression patterns of PagERF genes in different tissues of P. alba × P. glandulosa, including axillary buds, young leaves, functional leaves, cambium, xylem, and roots, and the results indicated that PagERF genes are expressed in all tissues of P. alba × P. glandulosa, especially in roots. Quantitative verification results were consistent with transcriptome data. When P. alba × P. glandulosa seedlings were treated with 6% polyethylene glycol 6000 (PEG6000), the results of RT-qRCR showed that nine PagERF genes responded to drought stress in various tissues. This study provides a new perspective on the roles of PagERF family members in regulating plant growth and development, and responses to stress in P. alba × P. glandulosa. Our study provides a theoretical basis for ERF family research in the future.


Introduction
Transcription factors are located in the nucleus and play important roles in regulating plant growth, development and abiotic stress response [1]. They are divided into families according to their specific conserved protein domains, such as Apetala2/ethylene responsive factor (AP2/ERF), myeloblastosis (MYB), [NAM (No apical meristem)/ATAF1/2 (Arabidopsis transcription activation factor 1/2)/CUC2 (cup-shaped cotyledon 2)] (NAC), basic region/leucine zipper (bZIP), etc. [2]. AP2/ERF is the largest transcription factor superfamily in plants and is found in many species, such as Bryum argenteum [3], Fagopyum tataricum [4], Prunus mume [5], and Phyllostachys edulis [6]. The AP2/ERF superfamily is divided into Apetala2 (AP2), Ethylene Responsive Factor (ERF), Related to Abscisic Acid Insensitive 3/Viviparous 1 (RAV), and soloist families according to the number and sequence similarity of AP2 domains [7]. The AP2 family contains two conserved AP2 domains, and its regulatory role in flower [8] and seed [9] development is widely reported. provides a theoretical basis for future research on the mechanisms of PagERF regulation of plant growth and development and responses to drought in P. alba × P. glandulosa.

Results
2.1. Identification of the PagERF Transcription Factor Family in P. alba × P. glandulosa To identify members of the PagERF family of transcription factors in P. alba × P. glandulosa, we compared protein sequences from P. alba × P. glandulosa with sequences representing AtAP2/ERF superfamily proteins in Arabidopsis thaliana; protein sequences with sequence identity (percentage identity) greater than 80% were considered as candidate sequences. According to the characteristics of ERF family protein sequences, we identified 209 PagERF proteins containing only one AP2 domain through conserved domain analysis (Figure 1), named PagERF1A to PagERF209B. We analyzed the following molecular properties of these protein sequences: number of amino acids, molecular weight, theoretical pI, instability index, aliphatic index, grand average of hydropathicity, and subcellular localization (Supplementary Table S1). The longest protein, PagERF206B, comprised 762 amino acids and its subcellular localization was in the nucleus. The shortest proteins, PagERF84A (molecular weight: 10,264.9 kDa) and PagERF183B (molecular weight: 10,278.93 kDa), each comprised 89 amino acids and by use of the Plant-mPLoc tool were found to be located in both the cytoplasm and the nucleus (Supplementary Table S1).
relationships, chromosome location, and gene structure. In addition, we used transcriptome data from axillary buds, young leaves, functional leaves, cambium, xylem, and roots of P. alba × P. glandulosa to analyze the tissue specificity of PagERF genes and used 6% PEG6000 to simulate drought stress. This study therefore provides a theoretical basis for future research on the mechanisms of PagERF regulation of plant growth and development and responses to drought in P. alba × P. glandulosa.

Identification of the PagERF Transcription Factor Family in P. alba × P. glandulosa
To identify members of the PagERF family of transcription factors in P. alba × P. glandulosa, we compared protein sequences from P. alba × P. glandulosa with sequences representing AtAP2/ERF superfamily proteins in Arabidopsis thaliana; protein sequences with sequence identity (percentage identity) greater than 80% were considered as candidate sequences. According to the characteristics of ERF family protein sequences, we identified 209 PagERF proteins containing only one AP2 domain through conserved domain analysis (Figure 1), named PagERF1A to PagERF209B. We analyzed the following molecular properties of these protein sequences: number of amino acids, molecular weight, theoretical pI, instability index, aliphatic index, grand average of hydropathicity, and subcellular localization (Supplementary Table S1). The longest protein, PagERF206B, comprised 762 amino acids and its subcellular localization was in the nucleus. The shortest proteins, PagERF84A (molecular weight: 10,264.9 kDa) and PagERF183B (molecular weight: 10,278.93 kDa), each comprised 89 amino acids and by use of the Plant-mPLoc tool were found to be located in both the cytoplasm and the nucleus (Supplementary Table S1).

Chromosomal Distribution of PagERF Genes
The hybrid poplar P. alba × P. glandulosa possesses two subgenomes of 19 chromosomes, subgenome A and subgenome B. Identifying the distribution of PagERF genes on chromosomes of P. alba × P. glandulosa (Supplementary

Chromosomal Distribution of PagERF Genes
The hybrid poplar P. alba × P. glandulosa possesses two subgenomes of 19 chromosomes, subgenome A and subgenome B. Identifying the distribution of PagERF genes on chromosomes of P. alba × P. glandulosa (Supplementary Table S2)

Phylogenetic Analysis of PagERF Proteins
To analyze the phylogenetic relationships of PagERF transcription factors, we reconstructed the phylogenetic tree of the PagERF proteins identified (Supplementary Table S3). We then divided the PagERF family into 10 subgroups (Figure 3), Classes I to X, according to their evolutionary relationships. Class I contained the fewest proteins, PagERF139B and PagERF206B, which belong to subgenome B; Class VIII contained five PagERF proteins, PagERF23A, PagERF24A, PagERF25A, PagERF26A, and PagERF27A, all belonging to subgenome A. Members of the other subgroups were associated with subgenomes A and B; Class IV and Class V were the largest subgroups, containing 39 members, followed by Class VI and Class X, containing 34 and 33 members, respectively.

Phylogenetic Analysis of PagERF Proteins
To analyze the phylogenetic relationships of PagERF transcription factors, we reconstructed the phylogenetic tree of the PagERF proteins identified (Supplementary Table S3). We then divided the PagERF family into 10 subgroups (Figure 3), Classes I to X, according to their evolutionary relationships. Class I contained the fewest proteins, PagERF139B and PagERF206B, which belong to subgenome B; Class VIII contained five PagERF proteins, PagERF23A, PagERF24A, PagERF25A, PagERF26A, and PagERF27A, all belonging to subgenome A. Members of the other subgroups were associated with subgenomes A and B; Class IV and Class V were the largest subgroups, containing 39 members, followed by Class VI and Class X, containing 34 and 33 members, respectively.

Localization and Duplication of PagERF Genes
To further explore the relationships among PagERF genes, we performed collinearity analysis on the replication events within the PagERF family. Eighteen gene pairs distributed on chromosome 2A and chromosome 5A showed the strongest relationships in subgenome A, while most of the other chromosomes had only one gene pair ( Figure 4A). We also identified 25 gene pairs distributed on chromosome 1B and chromosome 3B, chromosome 4B and chromosome 7B, and chromosome 5B and chromosome 7B in subgenome B ( Figure 4B). There were 38 gene pairs showing close relationships between chromosomes of subgenome

Localization and Duplication of PagERF Genes
To further explore the relationships among PagERF genes, we performed collinearity analysis on the replication events within the PagERF family. Eighteen gene pairs distributed on chromosome 2A and chromosome 5A showed the strongest relationships in subgenome A, while most of the other chromosomes had only one gene pair ( Figure 4A). We also identified 25 gene pairs distributed on chromosome 1B and chromosome 3B, chromosome 4B and chromosome 7B, and chromosome 5B and chromosome 7B in subgenome B ( Figure 4B). There were 38 gene pairs showing close relationships between chromosomes of subgenome A and subgenome B ( Figure 4C), with PagERF genes displaying strong similarity on chromosomes 5A and 5B between the two subgenomes. According to the Ka and Ks value, the ratio of Ka/Ks between gene pairs was less than one except for PagERF3A and PagERF119B (Supplementary Tables S4-S6). In summary, the duplication events affecting PagERF genes in P. alba × P. glandulosa are complex, including events on chromosomes within subgenome A, within subgenome B, and between subgenome A and subgenome B. mosomes 5A and 5B between the two subgenomes. According to the Ka and Ks value, the ratio of Ka/Ks between gene pairs was less than one except for PagERF3A and PagERF119B (Supplementary Tables S4-S6). In summary, the duplication events affecting PagERF genes in P. alba × P. glandulosa are complex, including events on chromosomes within subgenome A, within subgenome B, and between subgenome A and subgenome B.

Analysis of PagERF Gene Structure and PagERF Motif Composition
To further analyze the function of the PagERF family, we used the MEME tool to analyze the structure of PagERF genes and the protein motifs of the transcription factors ( Figure  5). The E-value of a motif is based on its log likelihood ratio, width, sites, background letter frequencies, and the size of the training set. The gene structure of PagERF genes showed very complex characteristics; PagERF31A had the longest intron, PagERF206B contained the largest number of introns, and most PagERF genes contained no introns. PagERF proteins in the same subgroup in the phylogenetic tree had similar motifs, which we named motif 1 to motif 10 according to their E values from low to high, and the protein family membership of all ten motifs was analyzed using the InterPro tool and found to be ERF factor, of which motif 1 and motif 2 both enriched the ethylene-activated signaling pathway (GO:0009873) (Supplementary Table S7). We obtained the following findings: (Ⅰ) The PagERF proteins containing motif 4 belonged to Class V, the PagERF proteins containing motif 3 belong to Class VI, and most of the Class IX PagERF proteins contained motifs 5, 2, 1, 8, and 6. (Ⅱ) Most of the PagERFs in Class II contained motifs 6, 5, 2, 1, 9. We identified 10 motifs among all PagERF protein sequences, with each gene encoding from one to six motifs. (Ⅲ) PagERF108A contained one motif, and PagERF22A and most PagERF proteins in Class V each contained six motifs.

Analysis of PagERF Gene Structure and PagERF Motif Composition
To further analyze the function of the PagERF family, we used the MEME tool to analyze the structure of PagERF genes and the protein motifs of the transcription factors ( Figure 5). The E-value of a motif is based on its log likelihood ratio, width, sites, background letter frequencies, and the size of the training set. The gene structure of PagERF genes showed very complex characteristics; PagERF31A had the longest intron, PagERF206B contained the largest number of introns, and most PagERF genes contained no introns. PagERF proteins in the same subgroup in the phylogenetic tree had similar motifs, which we named motif 1 to motif 10 according to their E values from low to high, and the protein family membership of all ten motifs was analyzed using the InterPro tool and found to be ERF factor, of which motif 1 and motif 2 both enriched the ethylene-activated signaling pathway (GO:0009873) (Supplementary Table S7). We obtained the following findings: (I) The PagERF proteins containing motif 4 belonged to Class V, the PagERF proteins containing motif 3 belong to Class VI, and most of the Class IX PagERF proteins contained motifs 5, 2, 1, 8, and 6. (II) Most of the PagERFs in Class II contained motifs 6, 5, 2, 1, 9. We identified 10 motifs among all PagERF protein sequences, with each gene encoding from one to six motifs. (III) PagERF108A contained one motif, and PagERF22A and most PagERF proteins in Class V each contained six motifs.

Analysis of Cis-Acting Elements in PagERF Promoters
We used PlantCARE to analyze the PagERF promoters (Supplementary Table S9). Functional annotation revealed a total of 21 functional elements ( Figure 6). Response elements for hormones including gibberellic acid, methyl jasmonate, and abscisic acid and auxin cis-acting elements, such as ABRE, ERE, of which there were eleven ABRE in the Pag-ERF150B promoter. In addition, the promoters also contained a variety of cis-acting elements that respond to abiotic stresses including drought, salt, and low temperature, including MYB binding sites involved in drought-inducibility, such as ARE, DRE, myc. Surprisingly, most PagERF promoters had myc and the promoter of PagERF16A, PagERF17A, PagERF18A, PagERF20A, PagERF21A, and PagERF131B, PagERF132B, PagERF133B, PagERF137B, Pag-ERF138B had ten myc in all (Supplementary Table S8). We also found cis-acting regulatory elements that were root specific, providing a research direction and theoretical basis for us to further explore the expression patterns of PagERF genes in different tissues of P. alba × P. glandulosa and their functions in response to abiotic stress.

Analysis of Cis-Acting Elements in PagERF Promoters
We used PlantCARE to analyze the PagERF promoters (Supplementary Table S9). Functional annotation revealed a total of 21 functional elements ( Figure 6). Response elements for hormones including gibberellic acid, methyl jasmonate, and abscisic acid and auxin cis-acting elements, such as ABRE, ERE, of which there were eleven ABRE in the PagERF150B promoter. In addition, the promoters also contained a variety of cis-acting elements that respond to abiotic stresses including drought, salt, and low temperature, including MYB binding sites involved in drought-inducibility, such as ARE, DRE, myc. Surprisingly, most PagERF promoters had myc and the promoter of PagERF16A, PagERF17A, PagERF18A, PagERF20A, PagERF21A, and PagERF131B, PagERF132B, PagERF133B, PagERF137B, PagERF138B had ten myc in all (Supplementary Table S8). We also found cis-acting regulatory elements that were root specific, providing a research direction and theoretical basis for us to further explore the expression patterns of PagERF genes in different tissues of P. alba × P. glandulosa and their functions in response to abiotic stress.

Expression Pattern Analysis of PagERF Genes
To explore the function of PagERF transcription factors in P. alba × P. glandulosa, we used RNA-seq data to analyze the expression patterns of PagERF genes in six different tissues: axillary bud, young leaf, functional leaf, cambium, xylem, and root ( Figure 7A). PagERF genes had specific expression patterns in different tissues ( Figure 7B, Supplementary Table S10). For example, most PagERF genes were strongly expressed in roots and weakly expressed in young leaves. In addition, PagERF174B, PagERF95A, PagERF66A, PagERF139B, and PagERF29A were strongly expressed in axillary buds, and PagERF130B was strongly expressed in the functional leaf. PagERF206B and PagERF189B were highly expressed in cambium, and PagERF135B, PagERF164B, and PagERF57A were highly expressed in xylem. These results indicated that PagERF genes are expressed in all tissues of P. alba × P. glandulosa, especially in roots.  and PagERF29A were strongly expressed in axillary buds, and PagERF130B was strongly expressed in the functional leaf. PagERF206B and PagERF189B were highly expressed in cambium, and PagERF135B, PagERF164B, and PagERF57A were highly expressed in xylem. These results indicated that PagERF genes are expressed in all tissues of P. alba × P. glandulosa, especially in roots.

RT-qPCR Validation of PagERF Gene Expression Patterns
To verify the accuracy of transcriptome data, we randomly selected 12 PagERF genes for quantitative validation (Figure 8). The quantitative results were consistent with the RNA-seq results. Consequently, most of the genes were more strongly expressed in roots than other tissues, especially PagERF198B and PagERF28A. PagERF40A also showed strong expression in cambium, and PagERF162B had relatively strong expression in axillary buds. In addition, Pag-ERF42A, PagERF103A, PagERF151B, and PagERF185B were substantially expressed in all tissues sampled. This indicated that PagERF genes have specific expression patterns in tissues of P. alba × P. glandulosa, especially in roots, which provided a basis for us to explore the expression patterns of PagERF genes in roots in response to drought.

RT-qPCR Validation of PagERF Gene Expression Patterns
To verify the accuracy of transcriptome data, we randomly selected 12 PagERF genes for quantitative validation (Figure 8). The quantitative results were consistent with the RNA-seq results. Consequently, most of the genes were more strongly expressed in roots than other tissues, especially PagERF198B and PagERF28A. PagERF40A also showed strong expression in cambium, and PagERF162B had relatively strong expression in axillary buds. In addition, PagERF42A, PagERF103A, PagERF151B, and PagERF185B were substantially expressed in all tissues sampled. This indicated that PagERF genes have specific expression patterns in tissues of P. alba × P. glandulosa, especially in roots, which provided a basis for us to explore the expression patterns of PagERF genes in roots in response to drought.

Analysis of PagERF Genes in Response to Drought in Seedlings
We used 6% PEG6000 treatment to simulate drought stress in P. alba × P. glandulosa and explored the response of nine PagERF genes in five tissues (axillary bud, young leaf, functional leaf, stem, and root) at eight time points using RT-qPCR ( Figure 9, Supplementary  Figures S2-S5). Most PagERF genes responded to drought in the roots but showed specific expression patterns at different time points. For example, expression of PagERF162B and PagERF28A was substantially up-regulated, and relative expression levels reached a maximum after 3 h under 6% PEG6000 treatment; meanwhile, PagERF144B showed relatively up-regulated expression levels at 24 h. However, some PagERF genes showed a down-regulated expression trend after drought treatment; for example, PagERF185B showed down-regulated expression within 12 h. Figure 8. Quantitative validation of expression patterns for 12 PagERF genes in different tissues. The X-axis represents the different tissues. The left Y-axis indicates the FPKM value obtained by RNAseq, and the right Y-axis indicates relative gene expression levels analyzed by RT-qPCR. Bars indicate mean ± SE (n = 3) from three independent trials. * p < 0.05, ** p < 0.01.

Analysis of PagERF Genes in Response to Drought in Seedlings
We used 6% PEG6000 treatment to simulate drought stress in P. alba × P. glandulosa and explored the response of nine PagERF genes in five tissues (axillary bud, young leaf, functional leaf, stem, and root) at eight time points using RT-qPCR (Figure 9, Supplementary  Figures S2-S5). Most PagERF genes responded to drought in the roots but showed specific expression patterns at different time points. For example, expression of PagERF162B and PagERF28A was substantially up-regulated, and relative expression levels reached a maximum after 3 h under 6% PEG6000 treatment; meanwhile, PagERF144B showed relatively

Discussion
AP2/ERF is a transcription factor superfamily that plays an essential role in plant growth and development in response to abiotic stress, and the function of ERF transcription factors in P. alba × P. glandulosa has not been studied, especially their regulatory roles in plant growth, development and responses to drought stress.
In this study, we identified 209 ERF transcription factors in P. alba × P. glandulosa according to their AP2 domain characteristics (Figure 1). Through phylogenetic analysis (Figure 3) and gene structure analysis ( Figure 5), we determined that PagERF genes in the same phylogenetic group with similar motif distribution had widely varying length and distribution of coding sequences. We used the two subgenomes of P. alba × P. glandulosa to analyze the chromosomal localization of PagERF transcription factor genes ( Figure 2, Supplementary Figure S1), which revealed complex intra-group and inter-group duplication relationships. In addition, we obtained eighteen gene pairs in subgenome A, 25 gene pairs in subgenome B, and 38 gene pairs between subgenome A and B using synteny analysis ( Figure  4), and the ratio of Ka/Ks indicated that P. alba × P. glandulosa might undergo multiple selecting evolutionary directions and the gene pair of PagERF3A and PagERF119B might play a crucial role in the evolution (Supplementary Tables S4-S6). These results indicate that Pag-ERF transcription factors may have similar functions in transcriptional regulation and interact closely. Owing to the particular characteristics of the P. alba × P. glandulosa genome [38], we speculate that the regulatory relationships among members of the PagERF family and other transcription factor families, such as MYB, in P. alba × P. glandulosa may be more complex than those in other poplars, including the molecular networks regulating plant growth and development or responding to abiotic stresses.

Discussion
AP2/ERF is a transcription factor superfamily that plays an essential role in plant growth and development in response to abiotic stress, and the function of ERF transcription factors in P. alba × P. glandulosa has not been studied, especially their regulatory roles in plant growth, development and responses to drought stress.
In this study, we identified 209 ERF transcription factors in P. alba × P. glandulosa according to their AP2 domain characteristics (Figure 1). Through phylogenetic analysis ( Figure 3) and gene structure analysis ( Figure 5), we determined that PagERF genes in the same phylogenetic group with similar motif distribution had widely varying length and distribution of coding sequences. We used the two subgenomes of P. alba × P. glandulosa to analyze the chromosomal localization of PagERF transcription factor genes ( Figure 2,  Supplementary Figure S1), which revealed complex intra-group and inter-group duplication relationships. In addition, we obtained eighteen gene pairs in subgenome A, 25 gene pairs in subgenome B, and 38 gene pairs between subgenome A and B using synteny analysis (Figure 4), and the ratio of Ka/Ks indicated that P. alba × P. glandulosa might undergo multiple selecting evolutionary directions and the gene pair of PagERF3A and PagERF119B might play a crucial role in the evolution (Supplementary Tables S4-S6). These results indicate that PagERF transcription factors may have similar functions in transcriptional regulation and interact closely. Owing to the particular characteristics of the P. alba × P. glandulosa genome [38], we speculate that the regulatory relationships among members of the PagERF family and other transcription factor families, such as MYB, in P. alba × P. glandulosa may be more complex than those in other poplars, including the molecular networks regulating plant growth and development or responding to abiotic stresses. ERF transcription factors play an important role in regulating plant growth and development, including the biosynthesis of secondary metabolites [39][40][41] and the biosynthesis and transduction of plant hormones [42], and mutual regulation with microRNA [43]. For example, chrysanthemum (Chrysanthemum morifolium) CmERF053 regulates the germination of shoot branches by regulating auxin and cytokinin transport in the axillary bud [18] and Arabidopsis AtERF6 represses leaf growth by inhibiting cell division and cell expansion in the leaf [44]. ERF transcription factors also modify stem growth and wood properties [35] and change stem elongation and secondary xylem lignification [45]. In addition, ERF transcription factors such as rice (Oryza sativa) OsERF71 influence root growth by changing root structure, causing formation of enlarged aerenchyma, and regulating genes related to cell wall thickening and lignin biosynthesis [46]. In this study, we found that most PagERF promoters contain CAT-box, O2 site, which was associated with plant growth and development; ERE, AuxRR, and the TCA element were cis-elements associated with phytohormone responsiveness (Figure 6). This showed that PagERF genes may be regulated to play a critical role in plant growth and development. Also, we analyzed the expression patterns of PagERF genes in different tissues of P. alba × P. glandulosa (Figures 7B and 8). This showed that most PagERF genes have high expression levels in roots, and a few genes are expressed in axillary bud, leaves, cambium, or xylem. Their expression patterns are relatively simple, being specifically expressed in certain tissues. For example, PagERF174B, PagERF95A, PagERF66A, PagERF139B, and PagERF29A show high expression levels in axillary buds; PagERF5A only displays high expression levels in young leaves; PagERF130B only shows high expression levels in functional leaves; PagERF206B and PagERFF189B have high expression levels only in cambium; PagERF135B, PagERF19A, PagERF164B, and PagERF57A display high expression levels only in xylem. Interestingly, regulation by ERF transcription factors has been reported in various plant tissues. Therefore, we hypothesize that PagERF transcription factors may specifically participate in the regulation of plant growth and development directly or indirectly in different tissues, especially roots, including plant hormone regulation and transcriptional regulation of binding to other transcription factors.
ERF transcription factors also play important regulatory roles in plant responses to abiotic stresses [47], including drought [48,49], cold [50,51], and salt [52,53]. In this study, we found that most PagERF promoters contain DRE elements, ABRE elements, and other elements involved in the response to stress ( Figure 6); some PagERF promoters also contained MYB binding sites associated with the response to drought. We used PEG treatment to simulate drought stress conditions for different periods and examined expression of nine PagERF genes in various tissues of P. alba × P. glandulosa in response to drought using RT-qPCR ( Figure 9, Supplementary Figures S2-S5). PagERF genes showed a strong response to drought in all tissues, especially roots. We speculate that the great changes in expression patterns of PagERF genes in response to abiotic stress may result from abnormal changes in plant hormone signals [54], such as gibberellins, cytokinins, and brassinosteroids, as well as MAPK signal transduction [55]. Moreover, homeostasis of the molecular network of transcriptional regulation in plants may be challenged under abiotic stress, affecting the transcriptional regulation process associated with PagERF proteins, further causing a series of defensive responses in various plant parts or organs. For example, differentiation of cambium cells and the growth process of xylem may be disturbed [56]; the role of PagERF proteins as growth factors regulating stem development may be affected, resulting in changes in stem growth and development. Accumulation of both proline and chlorophyll may be regulated by PagERF transcription factors, allowing control of H 2 O 2 content and stomatal conductance to resist drought stress in leaves [57]. Therefore, PagERF transcription factors play an important role in transcriptional regulation in the molecular network of P. alba × P. glandulosa in response to drought stress.
We conclude that PagERF transcription factors play a key regulatory role in the growth and development of P. alba × P. glandulosa and the molecular network in response to drought stress ( Figure 10). Taken together, these investigations benefit the selection of potentially improved PagERF transcription factors in the regulation of drought responses in P. alba × P. glandulosa and help to improve our understanding of the biological function of the ERF transcription factor family. However, in view of P. alba × P. glandulosa possessing two subgenomes, the interaction between PagERF transcription factors and their regulatory network relationships with other transcription factors or functional genes need to be further explored in the future. P. glandulosa and help to improve our understanding of the biological function of the ERF transcription factor family. However, in view of P. alba × P. glandulosa possessing two subgenomes, the interaction between PagERF transcription factors and their regulatory network relationships with other transcription factors or functional genes need to be further explored in the future.

Plant Materials and Treatments
Clonal wild-type P. alba × P. glandulosa (84K poplar) seedling tops with axillary buds and one single leaf were placed into ½-strength Murashige and Skoog (MS) medium supplemented with 0.1 mg/L indole-3-butyric acid (IBA) and 0.01 mg/L 1-naphthaleneacetic acid (NAA) and grown under long-day conditions (16-h light/8-h dark) at 23-25 °C for 20 days. For simulated drought stress, 20-day-old sterile seedlings were placed into 1/2 MS medium containing 6% (w/v) PEG6000 without exogenous hormones. Axillary buds, young leaves, functional leaves, stems, and roots were collected at 0, 3, 6, 12, 24, 48, 96, and 128 h from the onset of treatment and stored in liquid nitrogen for RNA extraction. At least three biological replicates were performed for each group.

Plant Materials and Treatments
Clonal wild-type P. alba × P. glandulosa (84K poplar) seedling tops with axillary buds and one single leaf were placed into 1 2 -strength Murashige and Skoog (MS) medium supplemented with 0.1 mg/L indole-3-butyric acid (IBA) and 0.01 mg/L 1-naphthaleneacetic acid (NAA) and grown under long-day conditions (16-h light/8-h dark) at 23-25 • C for 20 days. For simulated drought stress, 20-day-old sterile seedlings were placed into 1/2 MS medium containing 6% (w/v) PEG6000 without exogenous hormones. Axillary buds, young leaves, functional leaves, stems, and roots were collected at 0, 3, 6, 12, 24, 48, 96, and 128 h from the onset of treatment and stored in liquid nitrogen for RNA extraction. At least three biological replicates were performed for each group.

Chromosomal Localization and Evolutionary Analysis of PagERF Transcription Factors
Visual maps of 19 chromosomes from subgenomes A and B of P. alba × P. glandulosa based on General Feature Format (GFF) information in the TBtools software were analyzed, respectively, and replicated, duplicated, and orthologous pairs of PagERF genes were analyzed using MCScanX and Advanced Circos in TBtools software. The location of PagERF genes on chromosomes was analyzed using Gene Location Visualize from GTF/GFF in TBtools software.

Motif Analysis of PagERF Transcription Factors and Phylogenetic Tree Reconstruction
The conserved motifs of PagERF transcription factors were identified using MEME (http://meme-suite.org/tools/meme) (version 5.5.0) (accessed on 15 October 2022); 10 motifs were analyzed, and the acquiescent minimum and maximum width of motifs was set from 6 to 50. A phylogenetic tree was reconstructed using PagERF protein sequences identified by ClustalW algorithm in MEGA (version 7.0.26) software with the method of neighbor-joining algorithm and the parameters of pairwise deletion and 1000 replicates for bootstrap analysis. Interactive Tree of Life (iTOL) (https://itol.embl.de/) (version 6.6) (accessed on 15 October 2022) was used to further process the phylogenetic tree. Gene Structure View (Advanced) in TBtools software was used for visualization. The function of motifs was analyzed by InterPro (https://www.ebi.ac.uk/interpro/) (accessed on 15 October 2022).

Analysis of Cis-Acting Elements in PagERF Promoters
Regions 2000 bp upstream of PagERF coding sequences were selected as promoter sequences using the Gtf/Gff3 Sequences Extractor in TBtools software, and cis-acting elements of PagERF promoters were analyzed using PlantCARE (http://bioinformatics.psb. ugent.be/webtools/plantcare/html/) (version 1) (accessed on 15 October 2022). Simple BioSequence Viewer in TBtools software was used for visualization.

RNA Extraction and RT-qPCR
Total RNA from different plant material (axillary buds, young leaves, functional leaves, stems, cambium, xylem and roots) was extracted using a Qiagen RNeasy Plant Mini Kit (QIAGEN). DNA was digested using an RNase-Free DNase Set (QIAGEN) during the process of RNA extraction. Genes were selected for RT-qPCR analysis with specific primers (Supplementary Table S12). RT-qPCR primers were designed by Integrated DNA Technologies (IDT) database tools (https://sg.idtdna.com/Scitools/Applications/RealTimePCR/) (accessed on 15 October 2022), and primer specificities were tested by executing a Blastn search against local P. alba × P. glandulosa genome data. A total of 200 ng RNA was used for synthesizing cDNA using a PrimeScript RT reagent kit (TaKaRa); this cDNA was used for RT-qPCR analysis. Real-time PCR was performed on an Agilent M × 3000P Real-Time PCR System using a TB Green Premix ExTaq II (Tli RNaseH Plus) kit (TaKaRa). Three biological replicates and three technical replicates of each reaction were performed. The RT-qPCR procedure was as follows: pre-denaturation at 95 • C for 10 min and 40 cycles of 95 • C for 30 s, 60 • C for 1 min. Experimental data were processed using the 2 −∆∆CT method [58].

Conclusions
We identified members of the PagERF family in P. alba × P. glandulosa and analyzed the expression patterns of PagERF genes in axillary buds, young leaves, functional leaves, cambium, xylem, and roots, as well as expression patterns in response to drought stress. We characterized the conserved domains, physicochemical properties, gene structure, evolutionary relationships, and gene replication relationships on chromosomes of 209 PagERF transcription factors. Moreover, we used transcriptome data and quantitative RT-qPCR to verify the expression patterns of PagERF genes in different tissues of P. alba × P. glandulosa, showing that most PagERF genes are expressed strongly in roots. We observed the expression patterns of nine PagERF genes in different tissues in response to drought simulated using 6% PEG6000. This revealed that PagERF genes display different degrees of response to drought stress in various tissues. These results indicate that PagERF transcription factors play an important regulatory role in the molecular network of P. alba × P. glandulosa growth, development, and drought response.