Function and Evolution of C1-2i Subclass of C2H2-Type Zinc Finger Transcription Factors in POPLAR

C2H2 zinc finger (C2H2-ZF) transcription factors participate in various aspects of normal plant growth regulation and stress responses. C1-2i C2H2-ZFs are a special subclass of conserved proteins that contain two ZnF-C2H2 domains. Some C1-2i C2H2-ZFs in Arabidopsis (ZAT) are involved in stress resistance and other functions. However, there is limited information on C1-2i C2H2-ZFs in Populus trichocarpa (PtriZATs). To analyze the function and evolution of C1-2i C2H2-ZFs, eleven PtriZATs were identified in P. trichocarpa, which can be classified into two subgroups. The protein structure, conserved ZnF-C2H2 domains and QALGGH motifs, showed high conservation during the evolution of PtriZATs in P. trichocarpa. The spacing between two ZnF-C2H2 domains, chromosomal locations and cis-elements implied the original proteins and function of PtriZATs. Furthermore, the gene expression of different tissues and stress treatment showed the functional differentiation of PtriZATs subgroups and their stress response function. The analysis of C1-2i C2H2-ZFs in different Populus species and plants implied their evolution and differentiation, especially in terms of stress resistance. Cis-elements and expression pattern analysis of interaction proteins implied the function of PtriZATs through binding with stress-related genes, which are involved in gene regulation by via epigenetic modification through histone regulation, DNA methylation, ubiquitination, etc. Our results for the origin and evolution of PtriZATs will contribute to understanding the functional differentiation of C1-2i C2H2-ZFs in P. trichocarpa. The interaction and expression results will lay a foundation for the further functional investigation of their roles and biological processes in Populus.


Introduction
Zinc-binding domains were first identified in the protein TFIIIA of Xenopus oocytes, and function in the binding of IIA and DNA with cysteines and histidines in the enrichment folding domain centered on a zinc ion [1]. Zinc finger proteins (ZnFs) are the abundant proteins in eukaryotes, with diverse functions, including DNA recognition, RNA packaging, transcription regulation, protein folding, and assembly [2]. Structure studies of ZnFs revealed their diversity, which can be classified into classical, GATA, RanBP and A20 ZnFs according to their zinc ligation topology [3]. Classical ZnFs include a short β hairpin and α helix with a zinc atom coordinated with Cys-Cys-His-His or Cys-Cys-His-Cys subunits [3]. Most classical ZnF Cys2His2 zinc fingers are transcription factors that provide DNA binding and transcription regulation via their ββα framework of a Cys2His2 (C2H2) zinc finger module [2]. The stability of the C2H2 zinc finger ββα framework is maintained by the interlaced linkage provided by Zn 2+ , which binds with two pairs of histidines at the C-terminus of the α-helix and two cysteines at the end of the β-strand [4]. RS_CRZ1, a C2H2 ZnF of the tomato plant, is involved in the hostile environment encountered during Rhizoctonia solani host colonization [5].
The function of C2H2 ZnFs in growth regulation has been reported in plants. POPOVICH (POP), which encodes a C2H2 zinc-finger transcription factor (C2H2 ZTF), plays an important function during the evolution of Aquilegia nectar by regulating cell proliferation during

Phylogenetic Relationship of ZAT Proteins in P. trichocarpa
The sequences of 11 ZAT proteins in P. trichocarpa were obtained from Phytozome13 (https://phytozome-next.jgi.doe.gov/ (accessed on 20 January 2022)). To analyze the phylogenetic relationship between different ZATs in P. trichocarpa, their full-length protein sequences were aligned using ClustalW [24]. The phylogenetic tree of ZAT proteins was constructed using MEGA11 with the Maximum Likelihood (ML) method [25]. The bootstrap values reported for each branch reflected the percentage of 1000 replicate trees containing that branch.

Exon Intron Structure, Location, Conserved Motifs and Promoter in PtriZAT Genes
The exon intron structures of eleven PtriZAT genes were analyzed using the Gene Structure View package of TBtools (v1.098691) with geneID and gff3 data of P. trichocarpa [26]. The chromosomal location of all identified PtriZAT genes on the chromosome was mapped using Chromosome Map Tool Blast, Text merge for MCScanx, Quick run MCScanX Wrapper and circle gene view packages of TBtools (v1.098691). The conserved motifs in PtriZAT proteins were searched using Multiple Em for Motif Elicitation (MEME) (https://meme-suite.org/meme/tools/meme (accessed on 19 January 2022)). The conserved cis-elements in the promoters of PtriZAT genes were analyzed using the PLACE database (https://www.dna.arc.go.jp/PLACE/ (accessed on 19 January 2022)) with the 2 kb sequences upstream of the translational start codon.

Plant Materials and qRT-PCR Assays
The poplars were gathered from Kunming (E102.74 • , N25.17 • ), and well grown in the culture room of Southwest Forestry University, Kunming, with the cutting method and natural culture conditions. The fresh and healthy leaves, stems, and roots were collected. For drought, ABA, and salt treatment, there was a no-watering protocol (seven days), and 10 µM ABA and 250 Mm NaCl were added into the normal condition. Total RNAs were extracted using the RNAprep Pure Plant Plus Kit (Cat. DP441, Tiangen, Beijing, China) following the manufacturer's instructions for different tissues and stress treatment plant materials. RNA (1 µg) was used for reverse transcription with an EasyScript ® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR reagent Kit (Transgene, Beijing, China). The relative expression levels of individual genes were measured with gene-specific primers (Table S1) by real-time quantitative PCR (qRT-PCR) analysis, which was carried out in a 20 µL reaction mix with 1 µL of diluted cDNA template and TransStart ® Green qPCR SuperMix (Transgene, Beijing, China) with a Bio-Rad CFX96. The elongation factor 1 (EF1) served as the internal control [27].

Expression Profiles of PtriZAT Genes in Different Plants
P. trichocarpa and Physcomitrium patens transcriptome data were obtained from Phytozome 13 (https://phytozome-next.jgi.doe.gov/ (accessed on 20 January 2022)). The expression data of PtriZAT genes were used to analyze the expression profile of P. trichocarpa H1 genes at different development stages (female, male, bud, leaf, root, and stem) and treatments (ABA, ACC, BAP, BL, GA, NAA, SA, SL, and meJA). All the expression values demonstrated in the heatmap were calculated with log2 of the FPKM values of Phytozome 13. The heatmap of PtriZAT genes was obtained by Heml 1.0.3 [28].

Prediction of the Interaction Proteins of PtriZATs
The potential interaction proteins of PtriZATs were predicted using an online STRING server (https://string-db.org (accessed on 17 March 2022)) with the protein sequences of PtriZATs. All the interaction proteins of different PtriZATs were examined using contrastive analysis; the number and messages of corresponding proteins are listed in Table S2. The function annotation of interaction proteins was obtained in Phytozome 13.

Genome-Wide Identification of PtriZAT Members in P. trichocarpa
After a sequence similarity search in P. trichocarpa with the 20 C1-2i C2H2 ZTF members of A. thaliana, 17 proteins of P. trichocarpa were obtained (Table S3). Of the 17 candidate proteins, only 11 members that were conserved contained two ZnF_C2H2 domains, which was the specific feature of C1-2i subclass ZFs (Table 1, Figure 1A [18]). The subcellular localization of 11 PtriZATs proteins predicted by WOLF PSORT showed their nucleus location (https://wolfpsort.hgc.jp/ (accessed on 24 January 2022)). The length of PtriZATs proteins varied from 179 to 310 amino acids. The length of Zn-C2H2 domains also varied from 24 to 26 amino acids. All the PtriZATs were neutral or basic proteins, which were aliphatic with low hydropathicity (Table 1). To compare with other plants, we performed a homology search for typical plants using Arabidopsis ZATs through forward-and reverse-comparison, and confirmed the ZAT proteins with two Zn-C2H2 domains using the Web CD-Search Tool. Carya illinoinensis, Oryza sativa, Lactuca sativa, Manihot esculenta, Medicago truncatula, Solanum Tuberosum, and Zea mays contained more ZATs than P. trichocarpa. Ceratodon purpureus, β vulgaris L., Theobroma cacao, and Vitis vinifera had a small number of ZATs (Table S4).

Sequence Characteristics and Phylogenetic Relationships of PtriZATs
To obtain the sequence characteristic of PtriZATs, all 11 proteins were aligned and analyzed using BioEdit version 7.0.9.0 [29].
To understand the phylogenetic relationship of PtriZATs, a phylogenetic tree was built with sequences of PtriZAT proteins. The phylogenetic tree of PtriZATs revealed seven proteins that belonged to the same branches, consistent with the analysis results for protein structure (Figures 1 and 2A). The other four PtriZATs belonged to the other branch with longer footsteps. To analyze the phylogenetic relationship of PtriZATs with subclass C1-2i ZEPs of A. thaliana, an unrooted tree was constructed with their protein sequences. Except for the C1-2iD subgroup ZEPs, the other A. thaliana ZEPs were separated into an independent branch ( Figure S1). The phylogenetic tree of ZATs of a different plant also showed the two conserved subtypes of PtriZATs, which were sorted into two different branches with most ZATs of different plants, which may be according to different evolutionary relationships and sequence variation ( Figure S2). To understand the phylogenetic relationship of PtriZATs, a phylogenetic tree was built with sequences of PtriZAT proteins. The phylogenetic tree of PtriZATs revealed seven proteins that belonged to the same branches, consistent with the analysis results for protein structure (Figures 1 and 2A). The other four PtriZATs belonged to the other branch with longer footsteps. To analyze the phylogenetic relationship of PtriZATs with subclass C1-2i ZEPs of A. thaliana, an unrooted tree was constructed with their protein sequences. Except for the C1-2iD subgroup ZEPs, the other A. thaliana ZEPs were separated into an independent branch ( Figure S1). The phylogenetic tree of ZATs of a different plant also showed the two conserved subtypes of PtriZATs, which were sorted into two different branches with most ZATs of different plants, which may be according to different evolutionary relationships and sequence variation ( Figure S2).

Gene, Protein Structure, and Conserved Motifs of PtriZAT Genes
To further understand the structure of PtriZATs, we searched 15 conserved motifs in PtriZATs with the MEME software. As shown in Figure 2B, motif1, motif2, and motif3 were the most conserved motifs, which were identified in all the PtriZATs. Subgroup II PtriZATs had seven conserved motifs (motif 1, motif 2, motif 3, motif 4, motif 5, motif 6, and motif 8). PtriZATs of subgroup I can be classified into two subtypes, Potri.004G216900, Potri.006G121600, Potri.008G032300, and Potri.010G229400 were the first subtype, which conservedhad motif1, motif2, motif3 and motif5. Potri.010G209400, Potri.001G235800 and Potri.009G027700 were the second subtype, which when conserved had motif 1, motif 2, motif 3, motif 4 and motif 7. The PtriZATs of subgroup II were conserved and had motif 1, motif 2, motif 3, motif 4, motif 5, motif 6 and motif 8 (Table S5). Protein structure analysis of PtriZATs found two conserved ZnF-C2H2 domains, the locations of which differed according to the subgroups ( Figure 2C). The first subtype, Ptri-ZATs of subgroup I, had longer sequences, with ZnF-C2H2 domains near the C-terminal. Meanwhile, the second subtype, PtriZATs of subgroup I, had the shortest sequences. To reveal the coding characteristic of PtriZATs, the gene structure was investigated by comparing the CDS sequences. The results showed that all PtriZAT genes were conserved and

Gene, Protein Structure, and Conserved Motifs of PtriZAT Genes
To further understand the structure of PtriZATs, we searched 15 conserved motifs in PtriZATs with the MEME software. As shown in Figure 2B, motif1, motif2, and motif3 were the most conserved motifs, which were identified in all the PtriZATs. Subgroup II PtriZATs had seven conserved motifs (motif 1, motif 2, motif 3, motif 4, motif 5, motif 6, and motif 8). PtriZATs of subgroup I can be classified into two subtypes, Potri.004G216900, Potri.006G121600, Potri.008G032300, and Potri.010G229400 were the first subtype, which conservedhad motif1, motif2, motif3 and motif5. Potri.010G209400, Potri.001G235800 and Potri.009G027700 were the second subtype, which when conserved had motif 1, motif 2, motif 3, motif 4 and motif 7. The PtriZATs of subgroup II were conserved and had motif 1, motif 2, motif 3, motif 4, motif 5, motif 6 and motif 8 (Table S5). Protein structure analysis of PtriZATs found two conserved ZnF-C2H2 domains, the locations of which differed according to the subgroups ( Figure 2C). The first subtype, PtriZATs of subgroup I, had longer sequences, with ZnF-C2H2 domains near the C-terminal. Meanwhile, the second subtype, PtriZATs of subgroup I, had the shortest sequences. To reveal the coding characteristic of PtriZATs, the gene structure was investigated by comparing the CDS sequences. The results showed that all PtriZAT genes were conserved and had a single exon, except for Potri.006G121600, which contained two exons linked with a short intron and without UTR sequences ( Figure 2E).
Genes 2022, 13, x FOR PEER REVIEW 7 of 16 had a single exon, except for Potri.006G121600, which contained two exons linked with a short intron and without UTR sequences ( Figure 2E).

Cis-Elements in the Promoter Regions of PtriZAT Genes
To investigate the regulation pattern, we detected cis-elements in the promoter regions of PtriZAT genes ( Figure 2D, Table S6). Many cis-elements related to stress response and growth regulation were predicted by PlantCARE. The most frequent cis-elements were G-Box, which were detected in ten PtriZATs, except Potri.001G235800, which suggested the important function of light signals during transcription regulation. ABRE was the other widespread cis-elements in PtriZATs, which participated in the response of abscisic acid (ABA), which indicates the function of PtriZATs during stress response and growth regulation. The TGACG-motif, TC-rich repeats, LTR, AACA_motif, P-box, MBS, TCA-element, AuxRR-core, and WUN-motif, predicted in PtriZATs, function in response to MeJA, defense, low-temperatures, gibberellin, drought-inducibility, salicylic acid and auxin, which were important for plant growth under stress. Other growth regulation ciselements, such as circadian, CAT-box, RY-element, GC-motif, AT-rich sequence,

Cis-Elements in the Promoter Regions of PtriZAT Genes
To investigate the regulation pattern, we detected cis-elements in the promoter regions of PtriZAT genes ( Figure 2D, Table S6). Many cis-elements related to stress response and growth regulation were predicted by PlantCARE. The most frequent cis-elements were G-Box, which were detected in ten PtriZATs, except Potri.001G235800, which suggested the important function of light signals during transcription regulation. ABRE was the other widespread cis-elements in PtriZATs, which participated in the response of abscisic acid (ABA), which indicates the function of PtriZATs during stress response and growth regulation. The TGACG-motif, TC-rich repeats, LTR, AACA_motif, P-box, MBS, TCA-element, AuxRR-core, and WUN-motif, predicted in PtriZATs, function in response to MeJA, defense, low-temperatures, gibberellin, drought-inducibility, salicylic acid and auxin, which were important for plant growth under stress. Other growth regulation cis-elements, such as circadian, CAT-box, RY-element, GC-motif, AT-rich sequence, GCN4_motif, and MSA-like, were also detected, and involved in functions for circadian control, meristem expression, seed-specific regulation, anoxic specific inducibility, maximal elicitor-mediated activation, endosperm expression, and cell cycle regulation. In addition, a zinc metabolism regulation cis-element, O 2 -site, was detected in subgroup I PtriZATs, which implied the function of Zn 2+ in PtriZATs.

Expression Patterns of PtriZAT Genes in Different Tissues and Stress Treatment
To investigate the function of PtriZAT genes the transcript abundance of PtriZATs were analyzed based on the transcription data of four growth stages, nine stress treatments, and plants of two sexes from Phytozome 13 ( Figure S3). All PtriZATs were lowly expressed in all the growth stages and induced by stress treatment. The expression pattern of ten expressed PtriZATs was validated with qRT-PCR. The primer sequences for qRT-PCR are listed in Table S1. Based on the qRT-PCR analysis (Figure 4), the selected PtriZATs were highly induced by stress, especially ABA and salt. Notably, the significantly upregulated PtriZATs under abiotic stress, suggested their positive regulation, which may relate to the stress-response cis-elements (Figures 2 and 4). Except for stress treatment, the expression of most PtriZATs maintained a low level during different tissues. In addition, the expression of some PtriZATs varied in different tissues, which may be because of the growth-regulation cis-elements (Figures 2 and 4). At the same time, the expression pattern of some subgroup I PtriZATs (Potri.010G209400 and Potri.002G119300) were significantly induced by growth tissues such as root and stem compared to stress, which may relate to the functional complementarity caused by gene duplication events (Figures 3 and 4). Interestingly, a subgroup II PtriZAT (Potri.002G119300) highly induced both growth tissues and stress, which was different from the variation of the expression of other PtriZATs. The signal and process of PtriZATs involved need more research.
GCN4_motif, and MSA-like, were also detected, and involved in functions for circadian control, meristem expression, seed-specific regulation, anoxic specific inducibility, maximal elicitor-mediated activation, endosperm expression, and cell cycle regulation. In addition, a zinc metabolism regulation cis-element, O2-site, was detected in subgroup I Ptri-ZATs, which implied the function of Zn 2+ in PtriZATs.

Expression Patterns of PtriZAT Genes in Different Tissues and Stress Treatment
To investigate the function of PtriZAT genes the transcript abundance of PtriZATs were analyzed based on the transcription data of four growth stages, nine stress treatments, and plants of two sexes from Phytozome 13 ( Figure S3). All PtriZATs were lowly expressed in all the growth stages and induced by stress treatment. The expression pattern of ten expressed PtriZATs was validated with qRT-PCR. The primer sequences for qRT-PCR are listed in Table S1. Based on the qRT-PCR analysis (Figure 4), the selected Ptri-ZATs were highly induced by stress, especially ABA and salt. Notably, the significantly upregulated PtriZATs under abiotic stress, suggested their positive regulation, which may relate to the stress-response cis-elements (Figures 2 and 4). Except for stress treatment, the expression of most PtriZATs maintained a low level during different tissues. In addition, the expression of some PtriZATs varied in different tissues, which may be because of the growth-regulation cis-elements (Figures 2 and 4). At the same time, the expression pattern of some subgroup I PtriZATs (Potri.010G209400 and Potri.002G119300) were significantly induced by growth tissues such as root and stem compared to stress, which may relate to the functional complementarity caused by gene duplication events (Figures 3 and  4). Interestingly, a subgroup II PtriZAT (Potri.002G119300) highly induced both growth tissues and stress, which was different from the variation of the expression of other Ptri-ZATs. The signal and process of PtriZATs involved need more research.
To investigate the function of the interaction proteins of PtriZATs, we analyzed their cis-elements and expression patterns under different growth stages and stress. The specific interaction protein of Potri.010G209400, Potri.009G124900, was functional for growth regulation and all stress responses of P. trichocarpa. Potri.010G209400, a PtriZAT of subgroup I, functioned in a significant response of ABA, ACC, BAP, GA, NAA, SA, and meJA and may be related to Potri.009G124900 ( Figure 6, Table S8). Most interaction proteins had no explicit comments. The cis-elements with the 2000 bp upstream of these cis-elements included many stress-response binding motifs, such as MBS (MYB binding site involved in drought-inducibility), TATC-box (gibberellin responsiveness), LTR (low-temperature responsiveness), TCA-element (salicylic acid responsiveness), ABRE (abscisic acid responsiveness), ARE (anaerobic induction), AuxRR-core (auxin responsiveness), TGACG-motif (MeJA responsiveness), P-box (gibberellin responsiveness) and WUN-motif (wound responsiveness) (Figure 7, Table S9).

The Distribution of PtriZATs in Populus Species
To investigate the distribution of ZATs in different Populus species, ZATs with two conserved ZnF-C2H2 domains in different Populus species were obtained by BLASTP using PtriZATs. The results show that different ZATs were distributed in different Populus species, and 16 ZATs were in P. deltoids, 19 ZATs were in P. euphratica, 29 ZATs were in P. alba, and 38 ZATs were in P. tomentosa (Table S10). The sequence alignment of ZATs in different Populus species had variable subunits in conserved QAGGH ZnF-C2H2 domains ( Figure S4-S7, [18]). Total sequence variation of QAGGH was obtained in Po- Figure 7. Cis-elements of PtriZATs interaction proteins during the 2000 bp upstream sequences. The color labels were on the right, the cis-elements message seen on Table S9.

The Distribution of PtriZATs in Populus Species
To investigate the distribution of ZATs in different Populus species, ZATs with two conserved ZnF-C2H2 domains in different Populus species were obtained by BLASTP us-ing PtriZATs. The results show that different ZATs were distributed in different Populus species, and 16 ZATs were in P. deltoids, 19 ZATs were in P. euphratica, 29 ZATs were in P. alba, and 38 ZATs were in P. tomentosa (Table S10). The sequence alignment of ZATs in different Populus species had variable subunits in conserved QAGGH ZnF-C2H2 domains ( Figures S4-S7, [18] Figure S8). The other Populus ZATs were divided into two subgroups with PtriZATs ( Figure S8).

Characteristics of PtriZATs in P. trichocarpa
We identified eleven PtriZATs with two conserved ZnF_C2H2 domains in P. trichocarpa ( Figure 1, Table S3). Based on the phylogenetic tree of eleven PtriZATs, we classified them into two subgroups ( Figure 2). A comparison of the protein sequences and structures revealed the variation of PtriZATs. The length of the PtriZATs varied according to subgroups. The length of ZnF-C2H2 domains of subgroup I PtriZATs (25-27 amino acids) was longer than subgroup II (24-25 amino acids) ( Table 1). The location of ZnF-C2H2 domains in PtriZATs also varied according to the subgroups. The two ZnF-C2H2 domains were closer to the C-terminal in the first subtypes PtriZATs of subgroup I than subgroup II (Table 1, Figure 2). The length of spacing between the two ZnF-C2H2 domains varied from 19 to 54 amino acids, and the amino acid subunits also varied in different PtriZATs. The spacing between the core motifs of ZnF-C2H2 might be important for target sequence recognition [41], and there may be some difference in DNA-binding between different PtriZATs. The length of PtriZATs of subgroup I was also longer than subgroup II. Individual fingers of C2H2-ZF domains are specifically bound over a wide range of three base pair targets [42]. The higher quantity of ZnF-C2H2 domains and the ZnF-C2H2 DNA-binding landscapes confirmed the important role of C2H2 in the DNA binding of longer DNA sequences [43]. The length of ZnF-C2H2 domains of PtriZATs ensured the stability of a Zn finger and DNA binding ability, as reported previously [44]. The variety of spacers between the two fingers provided the necessary gaps between the core sites of the target DNA [18]. The highly conserved QALGGH motifs of PtriZATs were the same as Family C1 C2H2 of Arabidopsis and other plants, which were conserved zinc finger helices for DNA binding [18,41].

Different Distributions of ZATs in Populus Species
Different numbers of ZATs were obtained in different Populus (Table S10). The different distributions of ZATs in different Populus and plants might reflect their variation in function and evolution in Populus and plants (Tables S4 and S10). To investigate the evolutionary history of ZATs in different Populus and plants, a phylogenetic tree was built using protein sequences ( Figures S2 and S8). Seven P. deltoids ZATs, seven P. euphratica ZATs, five P. alba ZATs and fourteen P. tomentosa ZATs were separated into subgroup I with subgroup I PtriZATs. Nine P. deltoids ZATs, seven P. euphratica ZATs, six P. alba ZATs, seven P. tomentosa ZATs were separated into subgroup II with subgroup II PtriZATs. The separation of most Populus ZATs showed the conservation of ZATs in Populus. To investigate the difference in independent ZATs in Populus, sequence analysis was conducted using Populus ZATs. All the independent ZATs contained variation motifs in the Zn-C2H2 domain ( Figures S4-S7). C2H2 ZTFs, one of the largest gene families, were conserved in flora and fauna, the evolution of base-contacting residues for DNA binding, and the interaction of non-base-contacting residues with a DNA backbone, which plays a key role during their expansion in metazoans [45]. Sequence variation of QAGGH motifs obtained for different Populus species reflected the evolutionary events of Populus, especially the residue substitute of Gly ( Figures S4-S7). The hydrophobic amino acids surrounding the motifs of ZnF-C2H2 were involved in protein-protein interaction and might interact with other DNA-binding proteins [41,46]. The total sequence variation of QAGGH in Populus species might imply larger evolutionary events and different DNA-binding functions in Populus C2H2-ZnFs (Figures S4-S7).

Chromosomal Distribution and Gene Duplication of PtriZATs
According to the chromosome location results, Potri.004G216900, Potri.006G121600, and Potri.010G229400 were possibly derived from Potri.008G032300 (Figure 3). All four PtriZATs belonged to the first subtype of Subgroup I with conserved ZnF-C2H2 domains and amino acid subunits (Figures 1 and 2). The detected higher-expressed Potri.004G216900 under ABA treatment in the first subtype of subgroup PtriZATs may relate to the evolution and origin of these PtriZATs (Figure 4, Figure S3). Previous studies have identified that the Populus genome underwent at least three rounds of genome-wide duplication, followed by multiple segmental duplications, tandem duplications, and transposition events, such as retrotransposition and replicative transposition [30,47]. The origin of the four PtriZATs in the first subtype of subgroup ZATs may coincide with the genome-wide duplication. The location relationship of four Subgroup II PtriZATs was also a reflection of the genome-wide duplication, which was equalization pipeline-connected. However, the origins of other PtriZATs were unclear.

Functional Differentiation of PtriZATs
Most ZFPs take part in plant growth regulation and stress response. The ZFPs in G. hirsutum are involved in fiber cell growth and hormone response [48]. The faster and earlier response of PtaZFP2 monitored during stem bending and stress response implied its function in the growth and environmental adaption of Populus [49]. PeSTZ1, a C2H2-type zinc finger of P. euphratica, can regulate PeAPX2 to modulate ROS scavenging and enhance the freezing tolerance of Populus [13]. The higher expression of PtriZATs of subgroup II and the second subtype of subgroup I in all the stress treatments reflected the importance and widespectrum function of PtriZATs under stress (Figures 4 and S3, Table S5). The motifs and cis-elements related to phytohormone and abiotic stress resistance identified in PtriZATs indicated their stress resistance and growth regulation functions (Tables S6 and S7; [47]). Combining the expression level and cis-element analysis showed that PtriZATs take part in the ABA, meJA, and light responses of plants. The higher expression level of PtriZATs in male plants may imply a different function of PtriZATs during sex differentiation. In P. yunnanensis Dode, there were many sex-specific responses in growth regulation and physiological metabolism [50]. Under a stress condition, Populus also shows a sex-specific response in physiological, morphological, proteome, and gene expression levels to heavy metals, salinity, drought, and nutrient deficiency [51]. During growth and stress resistance, males of Populus were more adaptive [52]. The higher expression of PtriZATs in male Populus was consistent with the strength of male plants. The interaction proteins of PtriZATs support growth regulation and the stress response function, which may relate to epigenetic mechanisms, such as histone modification [33,34], cytosine methylation [38], and ubiquitination [40]. The highly induced Potri.006G192700 and Potri.016G045500 in male plants provided the specific function of PtriZATs during sex differentiation ( Figure 6, Table S8), which may be because of its function in the progression of meiosis in the male reproductive cell of SWI/SNF-Related proteins [39]. Cis-element interaction proteins contained and had a higher induced expression of PtriZATs genes, implying a function in the stress response of PtriZATs, which may occur through interaction with stress-related genes (Figures 4 and 7, Table S9). Except for the functional interaction proteins of PtriZATs reported in plants, the function of other interaction proteins with a role in animals and diseases needs more research in plants [53,54] (Table S2).
Overall, the study results indicate that the PtriZATs functions were the same as the functions for DNA recognition, transcription regulation, protein folding and assembly of ZnFs in eukaryotes [2], but the mechanisms in plants need further research.

Conclusions
This study provided a thorough overview of the C1-2i subclass of C2H2-type Zinc Finger of P. trichocarpa and Populus. It presented a new perspective on the evolution and function of PtriZATs. The phylogenetic analysis found that eleven PtriZATs were classified into two subgroups according to their sequence characteristics of the ZnF-C2H2 domain. The phylogenetic relationship, exon-intron structure, chromosomal location, conserved motifs, and cis-regulatory elements of PtriZATs were explored in this research. The phylogenetic relationship and exon intron structure analysis supported the two subgroups classification. Chromosomal mapping and collinearity analysis revealed the duplication events of PtriZATs. Furthermore, the cis-elements analysis and transcription data of PtriZATs revealed their special expression profiles focused on stress response and sex differentiation. The validation by qRT-PCR further illustrated the function of most PtriZATs under stress, especially ABA and salt. The variation in the expression pattern of different PtriZATs may relate to functional complementation and gene duplication. The interaction proteins of PtriZATs predicted using STRING were functional as regulators of epigenetic mechanisms, which may provide clues for the functional study of PtriZATs. Our study contributes to the understanding of the structure and role of growth tissues, PtriZATs under stress, and provides resources for further functional analysis in Populus. It may contribute to improving the stress resistance of Populus using molecular biology techniques such as overexpression/mutation and regulation of epigenetic modification in future research.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes13101843/s1, Figure S1: Phylogenetic analysis of ZAT proteins of A. thalina and P. trichorcarpa; Figure S2: Phylogenetic analysis of ZAT proteins from different plants; Figure S3: Expression of PtriZAT genes during different growth stage and stress response; Figure S4: Multiple sequence alignment of ZAT proteins in P. alba; Figure S5: Multiple sequence alignment of ZAT proteins in P. euphratica; Figure S6: Multiple sequence alignment of ZAT proteins in P. tomentosa; Figure S7: Multiple sequence alignment of ZAT proteins in P. deltoids; Figure S8: Phylogenetic analysis of ZAT proteins of different P. species; Table S1: The primer sequences used in the study; Table S2: The predicted network of protein-protein interactions of PtriZATs; Table S3: List of PrtriZATs in P. trichocarpa; Table S4: List of ZATs in different plants; Table S5: The conserved motifs identified in PtriZATs; Table S6: The cis-regulatory elements identified in the promoters of PtriZATs; Table S7: Expression data of ZATs of P. trichocarpa; Table S8: Expression data of interaction proteins of PtriZATs in P. trichocarpa; Table S9: The cis-regulatory elements identified in the promoters of interaction proteins of PtriZATs;

Conflicts of Interest:
The authors declare no conflict of interest.