Function and Characteristic Analysis of Candidate PEAR Proteins in Populus yunnanensis

PEAR proteins are a type of plant-specific DNA binding with one finger (Dof) transcription factors that play a key role in the regulation of plant growth, especially during phloem cell growth and seed germination in Arabidopsis. However, the identification, characteristics and function of PEAR proteins, particularly in woody plants, need to be further studied. In the present study, 43 candidate PEAR proteins harboring the conserved Zf-Dof domain were obtained in Populus yunnanensis. Based on phylogenetic and structural analysis, 10 representative PEAR candidates were selected, belonging to different phylogenetic groups. The functions of PEAR proteins in the stress response, signal transduction, and growth regulation of stem cambium and roots undergoing vigorous cell division in Arabidopsis were revealed based on their expression patterns as characterized by qRT-PCR analysis, in accordance with the results of cis-element analysis. In vitro experiments showed that the interaction of transcription factor (E2F) and cyclin indirectly reflects the growth regulation function of PEAR through light signaling and cell-cycle regulation. Therefore, our results provide new insight into the identity of PEAR proteins and their function in stress resistance and vigorous cell division regulation of tissues in P. yunnanensis, which may serve as a basis for further investigation of the functions and characteristics of PEAR proteins in other plants.


Introduction
Transcription factors play important roles in governing plant responses to the environment and regulating growth [1].The functions of several transcription factors are universal during plant growth and response [2][3][4].The DNA binding with one finger (Dof) family comprises plant-specific transcription factors, characterized by a single zinc finger structure, which were first found in maize [5,6].Dof proteins contain a conserved DNA-binding N-terminal and a transcriptional regulation C-terminal, and are found widely distributed in the plant kingdom.The conserved N-terminal contains a Cys2/Cys2 zinc finger DNA binding domain (Dof domain), which can specifically recognize AAAG sequences in the upstream region of target genes [7,8].
PEAR proteins are a kind of mobile Dof transcription factor that are enriched with cytokinin in the early protophloem sieve-element cells of Arabidopsis root procambial tissue and activate gene expression to promote radial growth [9].The following PEAR proteins have been characterized in Arabidopsis: PEAR1 (PHLOEM EARLY DOF 1, known as DOF2.4),PEAR2 (PHLOEM EARLY DOF 2, known as DOF5.1),DOF6, TMO6, OBP2, and HCA2 [9].Before the concept of PEAR proteins was understood, these PEAR members were reported to have important functions in regulating plant growth [9][10][11][12][13][14].Seed dormancy is an important mechanism of plant self-protection under unfavorable conditions.DNA-BINDING ONE ZINC FINGER 6 (DOF6) acts as a negative regulator of seed germination by forming complexes with downstream proteins (such as MLP329, TCP14, etc.) to regulate primary seed dormancy [10,11].The DELLA protein RGA-LIKE2 (RGL2) forms a complex with DOF6 that can active GATA12 (a gene that encodes GATA-type zinc finger transcription factor) to enforce primary seed dormancy [12].During plant growth, damage and wounding often occur due to biotic and abiotic factors [13].The Dof transcription factors HIGH CAMBIAL ACTIVITY2 (HCA2), TARGET OF MONOPTEROS6 (TMO6), DOF2.1, and DOF6 are activated at sites of wounding and cell wall damage to promote wound healing and tissue regeneration in Arabidopsis thaliana [13].Glucosinolates play a defensive role against herbivores and microorganisms and are important secondary metabolites in Capparales [15].AtDof1.1 (OBP2, a DOF transcription factor), is highly expressed in the vasculature of Arabidopsis, and is part of a regulatory network in glucosinolate biosynthesis [14].Cell division and differentiation are important plant responses during wound healing [16].The periclinal asymmetric cell division of interfascicular parenchyma cells is a basis for the formation of interfascicular cambium, which is important for the transport of water, nutrients, and signaling molecules in higher plants [17].HCA2 can regulate the formation of cambium and the development of vascular tissues in Arabidopsis through affecting periclinal asymmetric cell division [18].Although the concept of PEAR proteins was only recently proposed, the function of PEAR members have distributed in cell and tissue regulation [9].
The study of Dof transcription factors may provide clues for PEAR research.With the development of taxonomic studies, more Dof members with different classifications have been reported, such as five subfamilies found in wheat [19], nine classes in Chinese cabbage [20], four groups in Populus trichocarpa [21], four subgroups in pepper [22], nine groups representing four subfamilies in Jatropha curcas and Ricinus communis [23], four groups in Medicago truncatula [24], four classes constituting six clusters in tomato [25], four major clusters in Oryza sativa and Arabidopsis thaliana [26], and eight groups representing four subfamilies in Manihot esculenta [27].Their classification may give some clues for functional research.Dof is also distributed in various kinds of growth regulation processes in plants.For example, SIDOF10 regulates vascular tissue formation in the process of fruit setting, especially in young tissue undergoing vigorous cell division [28].OBP1, a Dof transcription factor in A. thaliana, is a transcriptional regulator of key cell cycle genes and is involved in the control of cell division via developmental signaling [29].SCAP1, a Dof transcription factor, regulates the expression of genes involved in stomatal functions and morphogenesis, which in turn regulate the essential processes of stomatal guard cell maturation [30].
PEAR is a kind of Dof transcription factor that contains characteristic Zf-Dof domains.In woody plants, Dof transcription factors have been identified in some species [21,31].But, the research on PEAR was lacking.Research is needed on the function and classification of PEAR, especially in woody plants, to explain their potential role in growth regulation in perennial plants.Populus yunnanensis, which is an important economic tree, is also important for ecological restoration, especially in the mining area of Southwest China [32,33].To identify and explore the functions of PEAR proteins in woody plants, we performed a homology comparison and characteristic analysis of Arabidopsis PEAR proteins and found 43 candidate PEAR proteins in P. yunnanensis.Then, we screened for PEAR proteins using enrichment data of P. trichocarpa (a poplar that has a close evolutionary relationship with P. yunnanensis), and intraspecific and interspecies collinearity.After the expression analysis and verification of interactions, we confirmed the functions of PEAR proteins in cell growth regulation and ABA response in P.yunnanensis.

Genome-Wide Identification of PEAR Candidates
After a sequence similarity search of P. yunnanensis with 6 Arabidopsis PEAR proteins, 43 candidate PEAR proteins were obtained (Table 1 and Table S1).All 43 P. yunnanensis candidate PEAR proteins contained conserved Zf-Dof domains [5].The length and molecular weight of P. yunnanensis PEAR proteins varied, but the Zf-Dof domains of these candidates were conserved and comprised 57-58 amino acids, except for Poyun21040, with 44 amino acids.Of the 43 candidate PEAR proteins, 14 candidates were weakly acidic and the other 29 candidates were neutral or basic.The subcellular location prediction of PEAR candidates revealed that the majority of PEAR candidates were localized in the nucleus, except for Poyun30052, Poyun30013 (mitochondria), and Poyun14051 (cytoplasm).

Sequence Characteristics and Phylogenetic Relationship of PEAR Candidates
To obtain the sequence characteristics of PEAR candidates in P. yunnanensis, all 43 proteins and 6 AtPEAR protein sequences were aligned and analyzed using BioEdit [35].All proteins contained a conserved Zf-Dof domain (Table 1).Based on their phylogenetic relationships, the sequences appeared to be diverse (Figure 1A,B).Except for DOF6 and TMO6, homologous PEAR candidates were found for the other four AtPEAR proteins.Poyun31130 and Poyun37101 shared a common evolutionary branch with AtHCA2.Poyun31130 and Poyun02469 were conserved in all domain corners according to models built using Dof zinc finger protein templates (from Glycine max and Populus alba) (Figure 1C,D).The amino acids of His72, Pro73, Gln74, Glu80, Thr82, and Pro129 in the domain helix of AtHCA2 in P. yunnanensis were found to have been replaced by amino acids with the same isoelectric point values.Poyun13901, Poyun29809, and Poyun14043 shared the same evolutionary branch as AtOBP2.The sequence alignment of AtOBP2 and its candidates revealed sequence diversity; even some domain-corner amino acids showed variation, such as Val131, Arg137, and Asn139, which were replaced by amino acids with the same physical and chemical properties.Poyun02469, Poyun38101, Poyun06788, Poyun17292, and Poyun22076 showed similarity with AtPEAR1 and AtPEAR2.The sequence similarity in the domain of AtPEAR1 and AtPEAR2 branch proteins exceeded 90%.

Phylogenetic Relationships and Molecular Characterization of PEAR Candidates in P. yunnanensis
The multiple sequence alignment of 43 PEAR candidates in P. yunnanensis was performed to construct a maximum likelihood phylogenetic tree.The tree revealed that the 43 PEAR candidates were classified into four main branches (Figure 2A).As shown in Figure 2, motif1, which is characteristic of Zf-Dof domain, was conserved and identified in all PEAR candidates (Figure 2B, Table S2).The AtHCA2 candidates (Poyun31130 and Poyun37101) contained conserved motifs 9, 10, and 19, but not motif 1.The AtOBP2 can-

Phylogenetic Relationships and Molecular Characterization of PEAR Candidates in P. yunnanensis
The multiple sequence alignment of 43 PEAR candidates in P. yunnanensis was performed to construct a maximum likelihood phylogenetic tree.The tree revealed that the 43 PEAR candidates were classified into four main branches (Figure 2A).As shown in Figure 2, motif1, which is characteristic of Zf-Dof domain, was conserved and identified in all PEAR candidates (Figure 2B, Table S2).The AtHCA2 candidates (Poyun31130 and Poyun37101) contained conserved motifs 9, 10, and 19, but not motif 1.The AtOBP2 candidates presented different motif distributions.Poyun29809 and Poyun14043 contained conserved motif 12, and motif 1 was not present in Poyun13901.The AtHCA2 candidates (Poyun31130 and Poyun37101) contained conserved motifs 9, 10, and 19 but not motif 1.The PEAR candidates contained conserved motifs 1, 7, 8, 10, and 17, and Poyun06788 also contained motif 11.Protein structure analysis of the AtHCA2 candidates revealed one conserved Zf-Dof domain, except Poyun21040, which contained a subfamily domain.The length of the proteins and the location of the domains were found to vary according to the phylogenetic tree branch (Figure 2C, Table 1).The length of AtPEAR1, AtPEAR2, AtOBP2 and AtHCA2 was in the middle of the range for all candidate proteins.The AtPEAR1 and AtPEAR2 candidates had longer N-terminal (more than 95 amino acids) than other candidates.The AtOBP2 candidates had a shorter N-terminal, which was less than 35 amino acids.The AtHCA2 candidates had an N-terminal of 75 amino acids.To reveal the coding characteristic of PEAR protein candidates, their gene structure was investigated by comparing their DNA sequences.The results showed that the gene structures varied according to the phylogenetic branch (Figure 2D).Only AtOBP2 candidates harbored one long intron, which separated the exon into two parts.The AtPEAR1 candidates harbored two introns and three exons.The AtPEAR2 candidates harbored a short intron.

Cis-Elements in Promoter Region of Candidate Proteins of AtPEAR Coding Genes
To investigate the regulatory mechanism, we searched for possible cis-elements in the promoter region of the candidate proteins of AtPEAR coding genes (Figure 2E, Table S3).Many of the cis-elements involved in hormone signaling, transcription regulation, and stress response were predicted using PLACE.The most frequent cis-elements found were those involved in light response, which suggests the important function of light signals.Ciselements related to transcription initiation, promoters, and enhancers revealed the strong transcriptional regulation capability of PEAR candidates.ABRE is another widespread cis-element involved in abscisic acid (ABA) response, which was indicative of the function of PEAR candidates in stress response and growth regulation [36].The presence of ARE, TGA-elements, TC-rich repeats, P-box, and TGACG-motifs, which are characteristic of cis-elements involved in hormone signaling, stress response and repair, reveals the function of PEAR candidates in anaerobic induction, auxin response, defense and stress response, gibberellin, MeJA response, and salicylic acid response [37][38][39].Other cis-elements involved in growth regulation, such as CAT-box, O2-site, MBS, G-box, LTR, circadian clock, and AT-rich elements, have also been detected, which are involved in the functions of meristem expression, zein metabolism regulation, MYB binding site of drought inducibility, MYB binding site of flavonoid light regulation, maximal elicitor-mediated activation, low-temperature response, and circadian rhythm [40,41].On the other hand, cis-elements involved in light response, transcription regulation, stress response, growth regulation, hormone signaling, and the binding site of AT-rich DNA binding protein (ATBP-1) were found to be enriched in the candidates AtOBP2, AtHCA2, and AtPEAR1.characteristic of PEAR protein candidates, their gene structure was investigated by comparing their DNA sequences.The results showed that the gene structures varied according to the phylogenetic branch (Figure 2D).Only AtOBP2 candidates harbored one long intron, which separated the exon into two parts.The AtPEAR1 candidates harbored two introns and three exons.The AtPEAR2 candidates harbored a short intron.

Expression Patterns of PEAR Candidates in Different Tissues and Stress Treatment
To investigate the function of PEAR candidates in poplar, we obtained publicly available expression data for 42 homologs of P. yunnanensis PEAR candidates in P. trichocarpa, which has the closest evolutionary relationship to P. yunnanensis (Figure S1).As shown in Figure 3, the expression of PEAR candidates varied in different tissues and treatments (Table S4).Poyun26623 (Potri.014G100900),Poyun32726 (Potri.017G084600),and Poyun22412 (Potri.008G087800)were significantly enriched under treatment with different kinds of hormones, and were linked with stress response cis-elements.Poyun36927 (Potri.012G063800),Poyun31438 (Potri.015G048300),Poyun30052 (Potri.007G036400),Poyun09599 (Potri.004G046600),Poyun21037 (Potri.011G055600),Poyun36479 (Potri.012G018700),and Poyun00813 (Potri.001G086400) were enriched in fast-growing tissues such as root tip and bud.The AtPEAR1 and AtPEAR2 candidates Poyun02469 (Potri.001G238400),Poyun38101 (Potri.011G140000),Poyun06788 (Potri.006G084200),Poyun17292 (Potri.010G205400),and Poyun22076 (Potri.008G055100) were enriched in young and fast-growing tissues (bud, stem, and root-tip).The AtHCA2 candidates were also characteristically enriched in fast-growing tissues, such as bud and stem node (Poyun37101 (Potri.012G081300))and during phytohormone treatment (Poyun31130 (Potri.015G077100)).The AtOBP2 candidates Poyun29809 (Potri.007G058200)and Poyun13901(Potri.005G149100)maintained low expression levels in all test samples; however, Poyun14043 was significantly enriched in fast-growing tissues such as bud, root tip, and stem.The commonality here is that all AtPEAR candidates were significantly enriched in fast-growing tissues.To verify whether candidate PEAR proteins would respond to stress treatment and were involved in growth regulation, we simulated stress conditions and isolated different tissues of P. yunnannesis.The relative expression of the representative candidate PEAR genes during stress response and tissue differentiation was further analyzed using qRT-PCR analysis (Figure 4).The PEAR candidates Poyun02469 and Poyun22076 were upregu- To verify whether candidate PEAR proteins would respond to stress treatment and were involved in growth regulation, we simulated stress conditions and isolated different tissues of P. yunnannesis.The relative expression of the representative candidate PEAR genes during stress response and tissue differentiation was further analyzed using qRT-PCR analysis (Figure 4).The PEAR candidates Poyun02469 and Poyun22076 were upregulated under stress treatment, especially under cold and ABA treatment (Figure 4A).On the other hand, Poyun02469 and Poyun22076 were significantly highly induced in the stem cambium, in contrast to the rest of the stem tissues and young roots (Figure 4B).The AtOBP2 candidates Poyun13901 and Poyun29809 responded negatively to drought and salt stress (Figure 4A).On the other hand, Poyun13901 and Poyun29809 were highly induced in the xylem, in the contrast to the stem tissues (Figure 4B).• C for 1 day; ABA: 50 µmol/mL abscisic acid (ABA) (dissolved in 50 mL water) were sprayed on plant material for 1 day.(B) Different tissues collected from leaves, epidermis cambium, xylem, and marrow were collected from two months old cutting seedlings.The 2 −∆∆CT method was used for calculated relative expression changes of selected PEAR genes, with respect to reference gene (EF1) [42].All qRT-PCR analyses were conducted with three replicates.Data represent means of three biological replicates and error bars indicate ± SE (n = 3).Asterisks indicate significant differences by Tukey's LSD test (* p < 0.05, ** p < 0.01).Significant relative to control in (A); significant contrast samples are connected by lines between tissues in (B).

Chromosomal Localization and Collinearity Analysis of PEAR Candidates
The chromosome distribution analysis of the P. yunnanensis genome revealed that the 43 PEAR candidates were unequally distributed among 18 chromosomes, except for chromosome (Chr) 16 (Table S5).Chr 3 and 5 contained the maximum number of PEAR candidates (four).The AtHCA2 candidate Poyun37101 was found to be located on Chr 18, and Poyun31130 was located on Chr 14. AtOBP2 candidates Poyun13901 and Poyun14043 were located on Chr 5, and Poyun29809 was located on Chr13.The AtPEAR candidates were distributed on Chr 1 (Poyun02469), Chr 2 (Poyun06788), Chr 6 (Poyun17292), Chr 9 (Poyun22076), and Chr 19 (Poyun38101).
Gene duplication events are always associated with plant evolution, which is one of the main phenomena underlying the expansion of gene families [43].Synteny analysis revealed that 36 PEAR candidates in P.yunnanensis demonstrated genomic synteny (Figure 5, Table S5).A total of 40 PEAR candidate pairs arose from genome duplication events.In the collinearity region of AtPEAR1 candidates (Poyun17292 and Poyun22076) and AtPEAR2 candidates (Poyun02469, Poyun38101, and Poyun06788), the repeated collinearity relationships found among them may have arisen from genome duplication events.There were 56 gene pairs between P.yunnanensis PEAR candidates and the A.thaliana genome.All AtPEAR candidates had more than one collinear gene in the A.thaliana genome, which indicates that there was conservation of PEAR between species and following genome duplication events of PEAR genes in P. yunnanensis.Poyun31130, an AtHCA2 candidate, exhibited collinearity with AtHCA2 (AT5G62940), providing evidence of the conservation of HCA2 across species.

Interaction Networks of PEAR Candidates
PEAR candidates are a type of Dof transcription factor that can form homodimers with other genes and proteins using their recognized DNA binding domain [13].The predicted protein-protein interaction network based on representative Populus PEAR candidates is shown in Figure 6.It was observed that most PEAR candidates interact with other transcription factors (Table S6), such as homeobox-leucine zipper and basic helix-loop-helix transcription factors.Aside from transcription factors, pectate lyase (which is related to the cell wall), DNAJ heat shock family proteins, flavin-binding kelch domain proteins, and kinesin-like proteins, were also predicted to interact with PEAR candidates.From the interaction results of candidate PEAR proteins, we could speculate that cell division and repair are important for their functioning.To further verify the protein-protein interactions of cell division proteins and PEAR candidates, we performed Y1H and Y2H experiments.The representative genes were cloned into experiment vectors.Unfortunately, we did not observe any direct interaction of these cell division proteins with PEAR candidates.During the experiment assessing protein interactions, we found that cyclin protein (Poyun15034) can directly interact with E2F transcription factors (Poyun05546 and Poyun12575) at the protein and gene level, possibly forming complexes with PEAR proteins to influence cell division (Figure 7).S5.

Interaction Networks of PEAR Candidates
PEAR candidates are a type of Dof transcription factor that can form homodimers with other genes and proteins using their recognized DNA binding domain [13].The pre-  S5.
interaction results of candidate PEAR proteins, we could speculate that cell division and repair are important for their functioning.To further verify the protein-protein interactions of cell division proteins and PEAR candidates, we performed Y1H and Y2H experiments.The representative genes were cloned into experiment vectors.Unfortunately, we did not observe any direct interaction of these cell division proteins with PEAR candidates.During the experiment assessing protein interactions, we found that cyclin protein (Poyun15034) can directly interact with E2F transcription factors (Poyun05546 and Poyun12575) at the protein and gene level, possibly forming complexes with PEAR proteins to influence cell division (Figure 7).

Discussion
Plant-specific Dof transcription factors are important for stress response, hormone signal regulation, and plant growth regulation [7,[28][29][30]44,45].As Dof transcription factors, PEAR proteins were found to be functional in early protophloem cells and cytokinin signaling [9].The typical characteristic of Dof is a single zinc finger structure containing a DNA-binding N-terminal and a transcriptional regulation C-terminal [6].Sequence similarity was the basis of protein research across plant species [26].A total of 43 candidate PEAR proteins in P. yunnanensis were obtained using BLAST (E-value of 1× 10 -5 ) with Arabidopsis PEAR proteins (Table S1) [9,26].In contrast to Arabidopsis (36 AtDofs) and rice (30 OsDofs), P. yunnanensis has more candidate PEAR proteins (Dof transcription factors) [26].Wheat (96 TaDofs) and Chinese cabbage (76 BraDofs) contained more Dof transcription factors than P. yunnanensis [19,20].As the same genus species, P. trichocarpa (41 PtrDofs) and P. yunnanensis (43 Dofs) contained different numbers of Dofs, which revealed the Dof transcription factors were various according to different species [21].The different amino acids lengths and physicochemical characteristics of the 43 candidate PEAR proteins revealed the differentiation (Table 1).To analyze the relationship between PEAR proteins and Dof transcription factors, we built a phylogenetic tree using protein sequences of candidate PEAR proteins in P. yunnanensis and Arabidopsis PEAR proteins (Figure 1A) [9].The physiological tree shows the evolutionary relationship and similarity between AtPEAR and P. yunnanensis PEAR candidates [46].The Zf-Dof domain common to 42 P. yunnanensis candidate PEAR proteins (except for poyun21040) verified the authenticity of the PEAR proteins (or Dof transcription factors) (Figure 1B) [5,6].
The homologs of six AtPEAR proteins were distributed in different groups with P.yunnanensis PEAR candidate proteins.Compared to AtHCA2, no homologs of AtDOF6 and AtTMO6 were found in P. yunnanensis.In Arabidopsis, AtDOF6 was regulator of primary seed dormancy [10].AtTMO6 was involved in cell wound healing and tissue regeneration after squeezing and cutting treatment [13].The missing homologs of AtDOF6 and AtTMO6 revealed their functional species was specific or focused on herbs with seed dormancy [10,11,13].AtHCA2 was also involved in tissue development through interfascicular cambium formation and vascular tissue development [18].The missing homologs of AtTMO6 may reveal functional complementation of AtTMO6 and AtHCA2 in P. yunnannesis according to their functional similarity in Arabidopsis and evolutionary similarity in P. yunnanensis (Figure 1A) [13].The sequence similarity was consistent with the phylogenetic tree (Figure 1B).Poyun02469, Poyun38101, Poyun06788, Poyun17292, and Poyun22076 share the same phylogenetic branch as AtPEAR1 and AtPEAR2, which share more sequence similarity compared to the other PEAR candidates (Figure 1A,B,D).Poyun31130 and Poyun37101 were found to be similar to AtHCA2 during their Zf-Dof domain structure, phylogenetic relation, protein characteristics, and substitution of amino acids (Table 1, Figure 1B,D).Poyun13901, Poyun29809, and Poyun14043 had phylogenetic similarity with AtOBP2, Poyun13901, and Poyun29809 exhibited more similar domain characteristics, phylogenetic relationships, and sequence similarity.All three AtOBP2 homolog PEAR candidates are basic proteins that contain identical amino acid substitutes.The sequence and structure similarity may indicate the functional similarity [47] and redundancy [48] between homologs.The homologs of AtHCA2 and AtPEAR contained similar protein building models (Dof) from different plant species (Glycine max and Populus alba), which indicated the variability between candidate PEAR proteins (Figure 1C,D) [49].On the other hand, the different similarity of P. yunnanensis PEAR homologs with Arabidopsis revealed the functional differentiation of Herbs and woody plants [21,50].
To verify the structure of and relationship between 10 representative PEAR candidates and other candidate proteins, a phylogenetic tree was constructed and characteristic analysis was performed with 43 candidate REAR protein (Dof transcription factor) sequences (Figure 2).Consistent with the 42 Dof transcription factors of P. trichocarpa, 43 candidate PEAR proteins with conserved Zn-Dof domains clustered into four groups based on their phylogenetic relationships and gene and protein structure (Figure 2) [21].Capsicum annuum L. [22], R. communis [23], M. truncatula [24], O. sativa [26], A. thaliana [26], and M. esculenta [27] also contained four groups of Dof transcription factors according to phylogenetic analysis, which showed the conserved classification.The phylogenetic relationship of PEAR candidates indicated that the AtOBP2 and AtPEAR homologs shared the same group and branch, but the conserved motifs and gene structure of the homologs varied, which implied the functional variability (Figure 2, Table S2) [49].The homologs of AtHCA2 were also found to differ from the homologs of AtOBP2 and AtPEAR in terms of phylogenetic relationship, conserved motifs, and gene structure (Figure 2).Their being on the same evolutionary branches verified the evolutionary similarity of the proteins; however, the evolutionary differentiation was predicted with different structure subunits [27].
Many cis-elements were predicted to be involved in plant growth and development, signal transduction, and stress response [51].The highly enriched cis-elements about light response, transcription enhancement, and ABRE revealed the function of PEAR candidates in plant growth regulation and stress response (Figure 2D, Table S3) [4,[36][37][38][39][40].To further speculate on the function of PEAR candidates, we collected the expression patterns of all PEAR candidate homologs of P. yunnanensis in P. trichocarpa (Figure 3, Table S4).Homologs of AtHCA2, AtOBP2, AtPEAR1, and AtPEAR2 were found to be highly enriched in fastgrowing tissues, especially in root tips and buds, which was consistent with their functions of tissue growth regulation in Arabidopsis (Figure 3, Table S4) [9,14,18].The high expression of homologs of AtPEAR and AtOBP2 in P. yunnanensis also verified their function on young tissue with rapidly separating cells (stem cambium and xylem), which correlates with their function of cell differentiation in Arabidopsis (Figure 4) [9,14].The unequal chromosome distribution of 43 PEAR candidates revealed their different and duplication functions (Table S5) [20].Collinearity analysis of the inter-chromosomal relationships of P. yunnanensis PEAR homologs revealed the gene duplication events of the PEAR candidate genes of P. yunnannesis, which were correlated with functional similarity (Figure 5A) [46].The collinearity relationship between different homologs of AtHCA2, AtPEAR1, and AtPEAR2 across species (Arabidopsis and P. yunnannesis) also revealed the duplication of different copies of genes with the same function and expression pattern, which may be correlated with functional redundancy (Figure 5B) [43].
The interaction of transcription factors and target genes or proteins is an important regulation mechanism of plant growth [52].Most of the P. yunnanensis candidate PEAR proteins were predicted to interact with other transcription factors and proteins (Table S6).Specifically, for the homologs of AtPEAR1, AtPEAR2, AtOBP2 and AtHCA2, common interaction proteins are bHLH transcription factor, DNAJ domain protein, flavin-binding kelch domain protein and kinesin-like protein (Figure 6).The bHLH transcription factors represented one of the largest families of transcription factors and are involved in biosynthesis, metabolism, transduction of plant hormones, and especially pleiotropic regulation in growth regulation and stress response [53].The interaction between P. yunnanensis PEAR candidates and bHLH transcription factors may be involved in regulation on young tissues and stress response (Figures 4 and 6).The function of DnaJ homolog genes (KAM2/GRV2) in the determination of embryonic growth axis in Arabidopsis was in accord with the high expression of PEAR candidates in young tissues with vigorous cell division (Figures 4 and 6) [54].FLAVIN-BINDING, KELCH REPEAT, F-BOX protein (FKF1) protein is essential for light signaling in Arabidopsis, and provides new information on growth regulation function of PEAR candidates in P. yunnanensis [55].The predicted interaction of the kinesin-like protein occurred during cell synchronization and cell-cycle regulation, which also provides evidence for the function on cell growth and differentiation regulation of P. yunnanensis PEAR candidates [56].Unfortunately, we did not observe a direct interaction between PEAR candidates and predicted proteins in the in vitro validation testing.However, the interaction between cyclin and E2F during the interaction testing of PEAR candidates revealed that the regulation and progression of the cell cycle are important for the function in growth regulation of P. yunnanensis PEAR candidates (Figure 7) [57,58].Our study found ten P. yunnanensis PEAR candidate proteins using phylogenetic and characteristic analysis.The expression and interaction results demonstrate that P. yunnanensis PEAR candidate proteins play a crucial role in stress response and growth regulation, especially in young tissues with vigorous cell division.This paper reveals the PEAR proteins in P. yunnanensis for further study.

Identification of Candidate PEAR Proteins
To identify candidate PEAR proteins in P. yunnanensis, a sequence similarity search was performed with Arabidopsis PEAR (AtPEAR) proteins using Basic Local Alignment Search Tool (BLAST) version ncbi-blast-2.7.1 (available at https://blast.ncbi.nlm.nih.gov/Blast.cgi;accessed on 23 March 2023) [9].The P. yunnanensis databases in BLAST were from the newly released sequencing data of NCBI (accession number PRJCA008692) [34].A total of 43 candidate PEAR proteins were obtained in P. yunnanensis based on their similarity to those in Arabidopsis (with E-value of 1e-5, calculated with sequence similarity by BLAST) [26].The protein sequences of the identified PEAR candidates in P. yunnanensis were subjected to domain analysis using PFAM (https://pfam.xfam.org/;accessed on 21 April 2023) and the Batch CD-Search Tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi;accessed on 21 April 2023).The features of protein domains were analyzed using Expasy-ProtParam (https://web.expasy.org/protparam/;accessed on 7 April 2023).The subcellular localization of the candidate PEAR genes was predicted using WOLF PSORT (https://wolfpsort.hgc.jp/;accessed on 7 April 2023).

Phylogenetic Analysis of PEAR Candidates in P. yunnanensis
The phylogenetic tree of AtPEAR proteins and candidate PEAR proteins of P. yunnanensis was constructed in MEGA11 (http://www.megasoftware.net/history.php;accessed on 21 April 2023) using the maximum likelihood (ML) method [59,60].The bootstrap value of each branch reflects the percentage of 1000 replicate trees containing that branch.The rooted Populus tree was obtained using OrthoFinder method with a phylogenetic inference of orthologs isolated from Populus species gene sequences, which were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/;accessed on 23 June 2022) [61].Sequence alignments results were obtained using Bioedit [62] with full candidate PEAR protein sequences, after multiple sequence alignment (1000 bootstraps) using ClustalW [63].

Gene Structure, Conserved Motifs, Domains and Cis-Elements of Candidate PEAR Genes
The gene structure of 43 candidate PEAR genes of P. yunnanensis was analyzed using the Gene Structure View package of TBtools (v1.098691) with gff3 data [34].The conserved motifs of PEAR candidates were detected using Multiple Em for Motif Elicitation (MEME) (https://meme-suite.org/meme/tools/meme; accessed on 31 March 2023).The conserved cis-elements in the promoters of candidate PEAR genes, corresponding to the 2 kb sequence upstream before the translational start codon, were detected using the PLACE database (https://www.dna.arc.go.jp/PLACE/; accessed on 31 March 2023).

Expression Pattern of PEAR Candidates in P. trichocarpa
Populus trichocarpa transcriptome data, obtained from Phytozome 13 (https://phytozomenext.jgi.doe.gov/;accessed on 5 May 2023), were used to analyze the expression profile of PEAR candidates in P. trichocarpa at different development stages (female and male plant leaves, swelling, early-dormant and fully open buds), organs (bud, leaf, root, root-tip, stem) and phytohormone treatments (ABA, ACC, BAP, BL, GA, NAA, SA, SL, and meJA).The expression values in the heatmap were calculated using the log2 of the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values from Phytozome 13.The heatmap of candidate PEAR genes of P. trichocarpa was obtained using HeatMap package of TBtool [64].

Plant Materials and qRT-PCR Assays
The cutting plant materials were collected from P. yunnanensis growing for one years at Kunming (E102.74• , N25.17 • ).Cuttings (about 25 cm) were cultured for two months in a green house (16 h light at 25 • C/8 h dark at 18 • C) of Southwest Forestry University with mixed nutrient medium pots (humus: quartz sand: perlite at 3:1:1) [65].For the different tissue tests, fresh healthy leaves, stems (including detached epidermis, cambium, xylem, and marrow), and roots were collected from two-month-old cutting seedlings.At least three biological replicates of plant tissues were immediately frozen in liquid nitrogen and stored in a refrigerator at −80 • C for drought testing, two days no-watering treatment were carried out (control: water once a day).A total of 50 µmol/mL abscisic acid (ABA) (dissolved in 50 mL water) was sprayed onto plant material for 1 day as ABA treatment.Plants grown in pots were cultured at 4 • C for 1 day as cold treatment.150 mM NaCl (dissolved in 100 mL water) was added to cultured plant pots for 1 day as salt treatment.At least three sets of biological replicates of treated P. yunnanensis leaves were collected for the test [66].
Total plant RNA was extracted using the RNAprep Pure Plant Plus Kit (Cat.DP441, Tiangen, Beijing, China) following the manufacturer's instructions, using different tissues and stress treatment materials.A total of 1 µg of RNA was used for reverse transcription using the EasyScript ® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR Reagent Kit (Transgene, Beijing, China).The relative expression levels of selected genes were measured using gene-specific primers (Table S7) and real-time quantitative PCR (qRT-PCR) analysis in a 20 µL reaction mix (TransStart ® Green qPCR SuperMix, Transgene, Beijing, China) with a Bio-Rad CFX96.The homolog of elongation factor 1 (EF1) in P. yunnanensis was used as the internal control [67].The 2 −∆∆CT method was used for relative expression changes of genes [42].All qRT-PCR analyses were conducted with three replicates.The statistically significant analysis used Tukey's LSD test (* p < 0.05, ** p < 0.01).

Location and Collinearity Analysis of PEAR Candidates Intraspecific and Interspecies
The chromosomal locations of the 43 candidate PEAR genes were mapped using Gene location visualize package in TBtools (v1.098691) [64].The collinearity analysis of candidate PEAR genes among P. yunnanensis (intraspecific) and between P. yunnanensis and A.thaliana (interspecies) were obtained using MCScanX Wrapper packages in TBtools (v1.098691) [64].

Prediction of the Interaction Proteins and Collinearity Analysis of PEAR Candidates in P. yunnanensis
The potential interaction proteins of P. yunnanensis PEAR candidate proteins were predicted using the STRING server (version 12.0, https://string-db.org;accessed on 7 April 2023) [68].Protein-protein interactions (PPIs) and functional annotation were analyzed using Populus alba as a background organism according to the sequence similarity in the database.All potential interacting proteins of PEAR in P. yunnanensis were collected according to the predicted interaction relationships in STRING, especially those predicted from curated databases and experiment.

Conclusions
This research presented a way to identify PEAR proteins in P. yunnanensis using phylogeny, structure, expression, and interaction verification analyses.Through phylogenetic and structural analysis, it was found that the PEAR candidates were divided into different groups, in which motifs were conserved within the same group, such as in AtPEAR1, AtPEAR2, AtOBP2, and AtHCA2, which can be used in the initial identification of PEAR proteins.The expression patterns and qRT-PCR analyses verified the function of PEAR candidates in growth regulation in tissues where there is vigorous cell division and signal response, further verifying the authenticity and functional conservation of PEAR candidates in poplar.The predictions of and experiments on protein interactions showed that PEARs are involved in regulating plant growth through cell-cycle regulation and light signaling.Therefore, our results provide valuable information regarding the identification of PEAR proteins and their function in poplar.

Figure 1 .
Figure 1.Sequence alignment and structure analysis of AtPEAR proteins and their candidates in P. yunnanensis.(A) Maximum likelihood phylogenetic tree of 6 AtPEAR and 43 PEAR candidate proteins.Amino acid sequences were aligned with Clustal W, and phylogenetic tree was constructed using MEGA 11. (B) Multiple sequence alignment of AtPEAR proteins and their candidates in P. yunnanensis with Bi-oEdit.Red underline and letters denote domains of PEAR proteins.Four representative cysteine residues of zinc-finger structure are indicated with red box.Black shading indicates > 90% in corresponding position.(C,D) Protein structure of Zf-Dof domain in representative PEAR candidate proteins in P. yunnanensis.Secondary structure of Poyun02469 (PEAR2 homolog) (C) and Poyun31130 (HCA homolog) (D) predicted using Expasy-ProtParam.The Poyun02469 model was built using a AOA4V6AAQ1.1.A template (Populus alba) (84.47% sequence identity, 1.00 coverage).The Poyun31130 model was built using a I1KCF8.1.A template (Glycine max) (80.26% sequence identity, 0.99 coverage).The arrows labeled amino acids in the model were conserved in the domain helix (His72, Pro73, Gln74, Glu80, Thr82 and Pro129, (C)) and domain corner (Val131, Arg137, and Asn139, (D)).

Figure 1 .
Figure 1.Sequence alignment and structure analysis of AtPEAR proteins and their candidates in P. yunnanensis.(A) Maximum likelihood phylogenetic tree of 6 AtPEAR and 43 PEAR candidate proteins.Amino acid sequences were aligned with Clustal W, and phylogenetic tree was constructed using MEGA 11. (B) Multiple sequence alignment of AtPEAR proteins and their candidates in P. yunnanensis with BioEdit.Red underline and letters denote domains of PEAR proteins.Four representative cysteine residues of zinc-finger structure are indicated with red box.Black shading indicates >90% in corresponding position.(C,D) Protein structure of Zf-Dof domain in representative PEAR candidate proteins in P. yunnanensis.Secondary structure of Poyun02469 (PEAR2 homolog) (C) and Poyun31130 (HCA homolog) (D) predicted using Expasy-ProtParam.The Poyun02469 model was built using a AOA4V6AAQ1.1.A template (Populus alba) (84.47% sequence identity, 1.00 coverage).The Poyun31130 model was built using a I1KCF8.1.A template (Glycine max) (80.26% sequence identity, 0.99 coverage).The arrows labeled amino acids in the model were conserved in the domain helix (His72, Pro73, Gln74, Glu80, Thr82 and Pro129, (C)) and domain corner (Val131, Arg137, and Asn139, (D)).

Figure 2 . 2 .
Figure 2. Phylogenetic tree, motifs, cis-elements, and structure of PEAR candidates in P. yunnanensis.(A) Phylogenetic tree constructed based on maximum likelihood method.(B) Conserved motifs Figure 2. Phylogenetic tree, motifs, cis-elements, and structure of PEAR candidates in P. yunnanensis.(A) Phylogenetic tree constructed based on maximum likelihood method.(B) Conserved motifs of PEAR candidates as predicted using Multiple Em for Motif Elicitation (MEME).A total of 15 conserved motifs are represented by colored boxes.(C) Schematic diagram of Zf-Dof domains in PEAR candidates; green boxes indicate Zf-Dof domains.(D) Gene structure of PEAR candidates.Yellow boxes indicate CDS, and green boxes indicate non-coding sequences.(E) Cis-elements of PEAR candidates within 2000 bp upstream sequences.

21 Figure 4 .
Figure 4. Expression patterns of four representative candidate PEAR genes by qRT-PCR.(A) Under stress treatment.CK: untreated control leaves; drought: two days no-watering treatment; salt: 150 mM NaCl (dissolved in 100 mL water) were added to cultured plant pots for 1 day; cold: seedlings grown in pots were cultured at 4 °C for 1 day; ABA: 50 µmol/mL abscisic acid (ABA) (dissolved in 50 mL water) were sprayed on plant material for 1 day.(B) Different tissues collected from leaves, epidermis cambium, xylem, and marrow were collected from two months old cutting seedlings.The 2 -ΔΔCT method was used for calculated relative expression changes of selected PEAR genes, with

Figure 4 .
Figure 4. Expression patterns of four representative candidate PEAR genes by qRT-PCR.(A) Under stress treatment.CK: untreated control leaves; drought: two days no-watering treatment; salt: 150 mM NaCl (dissolved in 100 mL water) were added to cultured plant pots for 1 day; cold: seedlings grown

Figure 5 .
Figure 5. Gene duplication events and collinearity of candidate PEAR genes of (A) P. yunnanensis and (B) between P. yunnanensis and A.thaliana.Collinearity pairs of candidate PEAR genes are connected with black lines.Chromosome numbers are indicated near each chromosome boxes.The collinearity pairs were list in TableS5.

Figure 5 .
Figure 5. Gene duplication events and collinearity of candidate PEAR genes of (A) P. yunnanensis and (B) between P. yunnanensis and A.thaliana.Collinearity pairs of candidate PEAR genes are connected with black lines.Chromosome numbers are indicated near each chromosome boxes.The collinearity pairs were list in TableS5.

Figure 6 .
Figure 6.Predicted network of protein-protein interactions of PEAR candidates of P. yunnanensis using STRING.Spheres represent interaction proteins.The orange proteins in the center represented ten P. yunnanensis PEAR proteins.Color lines represent different types of interaction relationships between proteins.Known interactions: interaction relationships verified by curated databases (blue) or experiments (purple); predicted interactions: interaction relationships predicted by gene neighborhood location (green), gene fusion (red), or gene co-occurrence (blue violet); others: possible interactions of proteins by text mining (olive), co-expression (black), or protein homology (gray-purple).

Figure 6 . 21 Figure 7 .
Figure 6.Predicted network of protein-protein interactions of PEAR candidates of P. yunnanensis using STRING.Spheres represent interaction proteins.The orange proteins in the center represented ten P. yunnanensis PEAR proteins.Color lines represent different types of interaction relationships between proteins.Known interactions: interaction relationships verified by curated databases (blue) or experiments (purple); predicted interactions: interaction relationships predicted by gene neighborhood location (green), gene fusion (red), or gene co-occurrence (blue violet); others: possible interactions of proteins by text mining (olive), co-expression (black), or protein homology (gray-purple).Int.J. Mol.Sci.2023, 24, x FOR PEER REVIEW 13 of 21

Table 1 .
Characteristics of PEAR candidates in P. yunnanensis.