The DBB Family in Populus trichocarpa: Identification, Characterization, Evolution and Expression Profiles

The B-box proteins (BBXs) encode a family of zinc-finger transcription factors that regulate the plant circadian rhythm and early light morphogenesis. The double B-box (DBB) family is in the class of the B-box family, which contains two conserved B-box domains and lacks a CCT (CO, CO-like and TOC1) motif. In this study, the identity, classification, structures, conserved motifs, chromosomal location, cis elements, duplication events, and expression profiles of the PtrDBB genes were analyzed in the woody model plant Populus trichocarpa. Here, 12 PtrDBB genes (PtrDBB1–PtrDBB12) were identified and classified into four distinct groups, and all of them were homogeneously spread among eight out of seventeen poplar chromosomes. The collinearity analysis of the DBB family genes from P. trichocarpa and two other species (Z. mays and A. thaliana) indicated that segmental duplication gene pairs and high-level conservation were identified. The analysis of duplication events demonstrates an insight into the evolutionary patterns of DBB genes. The previously published transcriptome data showed that PtrDBB genes represented distinct expression patterns in various tissues at different stages. In addition, it was speculated that several PtrDBBs are involved in the responsive to drought stress, light/dark, and ABA and MeJA treatments, which implied that they might function in abiotic stress and phytohormone responses. In summary, our results contribute to the further understanding of the DBB family and provide a reference for potential functional studies of PtrDBB genes in P. trichocarpa.


Introduction
Transcription factors (TFs) are important regulatory proteins that act on various stages of plant growth and development and regulate gene expression under stress conditions by activating or inhibiting the transcription of target genes [1,2].According to the data provided by PlantTFDB, the poplar genome encodes 2455 putative TFs and can be divided into 58 subfamilies.Moreover, zinc finger transcription factors are a large gene family of TFs, which can be divided into different subfamilies based on the motif structures and functional characteristics of each member [2,3].The B-box (BBX) protein belongs to a subfamily of the zinc finger TFs, which participate in transcriptional regulation by binding to other proteins or DNA [4].
The BBX transcription factor is characterized by the existence of one or two B-box domains at the N-end of its protein and CCT domains at the C-end [5,6].They are considered to be involved in the regulation of seedling photomorphogenesis, plant phototropism and flowering induction and affect hormone-signaling pathways [7,8].The double B-box (DBB) transcription factor carries two B-box domains (B-box1 and B-box2) in the N-terminal region without a CCT domain.Moreover, the length of each B-box is about 40 amino acids, and 8-15 amino acids are present between the two B-box domains [5,9].
Poplar is a model plant in woody plant research which grows rapidly and whose genes are easily studied.The Populus trichocarpa genome provides the possibility for gene identification and function and evolution analyses.The DBB gene family has been identified and described in some plants, such as maize [19,20], rice [9], Arabidopsis [5,21], bamboo [22], and Saccharum [23], but no systematic research or analysis of the DBB gene family in Populus have been conducted.In this study, 12 putative PtrDBB genes were identified and classified into four groups, taking A. thaliana, O. sativa, Z. mays, P. patens, S. moellendorffii, P. abies and A. trichopoda DBBs and the conserved domains as references [24].Synthesis and comprehensive bioinformatics analyses were performed to study their phylogenetic relationship, gene and protein structures, domain composition, and chromosome location promoter cis elements.Moreover, the expression patterns of 12 PtrDBB genes under abiotic stress (light/dark and drought) and phytohormone (ABA and MeJA) treatments were inspected in poplar.On the basis of the information obtained from this study, we provide a precious resource for comprehensive research on the functionality of the poplar DBB gene family.

Identification of PtrDBB Genes in Populus
To perform the identification of putative PtrDBB genes in the Populus genome, an extensive search and the alignment of Arabidopsis DBB proteins sequences were conducted by obtaining these sequences from the P. trichocarpa genomic database.After removing the redundant sequences, 12 PtrDBB genes (PtrDBB1-PtrDBB12) were identified, all of which were used to confirm the existence of the two conserved B-box domains without a CCT domain through genome-wide analysis.The domain structures of 12 poplar DBB proteins generated using NCBI and Batch CD-Search are shown in Figure S1.To provide a deeper understanding of the similarity within the poplar DBB gene family, we used MEGA-X 11.0.13 and JalView 2.11.3.0 to perform the multiple alignment of the domain sequences of 12 putative PtrDBBs and compared the conservative sequences of B-box1 and B-box2.As shown in Figure 1, the lengths of B-box1 and B-box2 were 38 aa and 36-38 aa, respectively, and the interval length of the two B-box domains was 15 aa.Moreover, the lengths of Box1 and Box2, as well as some differences between the groups, were generally conservative.In addition, details of the PtrDBB genes are exhibited in Table 1, including their gene ID, chromosome location, coding sequence length, protein length, physicochemical parameters, molecular weight (MW) and exon numbers.The length of the coding sequences varied from 555 bp (PtrDBB5) to 936 bp (PtrDBB6).The poplar DBB genes encoded amino acid sequences ranged from 185 to 311 aa, and the predicted molecular weight (MW) varied from 20.51 to 34.34 kDa.In addition, the theoretical isoelectric points ranged from 4.77 to 7.05.
all of which were used to confirm the existence of the two conserved B-box domains out a CCT domain through genome-wide analysis.The domain structures of 12 p DBB proteins generated using NCBI and Batch CD-Search are shown in Figure S1.T vide a deeper understanding of the similarity within the poplar DBB gene family, we MEGA-X 11.0.13 and JalView 2.11.3.0 to perform the multiple alignment of the do sequences of 12 putative PtrDBBs and compared the conservative sequences of B-box B-box2.As shown in Figure 1, the lengths of B-box1 and B-box2 were 38 aa and 36respectively, and the interval length of the two B-box domains was 15 aa.Moreove lengths of Box1 and Box2, as well as some differences between the groups, were gen conservative.In addition, details of the PtrDBB genes are exhibited in Table 1, incl their gene ID, chromosome location, coding sequence length, protein length, ph chemical parameters, molecular weight (MW) and exon numbers.The length of the c sequences varied from 555 bp (PtrDBB5) to 936 bp (PtrDBB6).The poplar DBB gen coded amino acid sequences ranged from 185 to 311 aa, and the predicted mole weight (MW) varied from 20.51 to 34.34 kDa.In addition, the theoretical isoelectric p ranged from 4.77 to 7.05.

Analysis of Phylogenetic Relationship and Gene Structures of the DBB Genes
To explore the phylogenetic relationship of the poplar DBB family, a neighbor-joining phylogenetic tree was generated with MEGA-X by aligning 12 PtrDBB protein sequences with 14, 6, 12, 6, 4, 2 and 3 protein sequences from Arabidopsis thaliana (dicotyledonous subfamily), Oryza sativa (monocotyledons subfamily), Zea mays (monocotyledons subfamily), Physcomitrella patens, Selaginella moellendorffii, Picea abies and Amborella trichopoda (dicotyledonous subfamily), respectively.Detailed information of the DBB genes from A. thaliana, O. sativa, Z. mays, P. patens, S. moellendorffii, P. abies and A. trichopoda are listed in Supplementary Table S1.Using this phylogenetic tree, the DBB gene family was classed into six subfamilies based on the evolutionary relationships and motif analysis of DBB proteins, and the PtrDBBs were also divided into four groups (G1, G3, G5 and G6) but not G2 and G4.The numbers of PtrDBB members in different groups varied; G1, G3, G5 and G6 contained four, two, two and four genes, respectively (Figure 2).
To gain further understanding of the phylogenetic relationship of PtrDBB genes, a separate phylogenetic tree was constructed only using the PtrDBB protein sequences, and the PtrDBB proteins were divided into four subfamilies, which is in accordance with the phylogenetic tree of A. thaliana, O. sativa, Z. mays, P. patens, S. moellendorffii, P. abies and A. trichopoda.To gain further information of the structural features of poplar DBB family members, the analysis of exon/intron organizations was performed by matching the PtrDBB genes' genomic DNA sequences with their cDNA sequences.Figure 3 displays the exon/intron predictions of all 12 PtrDBB genes.The number of exons in the four distant groups varied from two to six.PtrDBB10 had the highest quantities of exons (6), and PtrDBB3 and PtrDBB9 only contained two exons.However, highly similar exon/intron structures were shared in the majority of the PtrDBB genes that were assembled in the same group.For instance, two PtrDBBs contained three exons in G2 and G3, respectively, and the members within G4 had four or five exons.Overall, the phylogenetic relationship and conservated gene structure results provide a reliable basis for the classification of poplar DBB family members.liana, O. sativa, Z. mays, P. patens, S. moellendorffii, P. abies and A. trichopoda are listed in Supplementary Table S1.Using this phylogenetic tree, the DBB gene family was classed into six subfamilies based on the evolutionary relationships and motif analysis of DBB proteins, and the PtrDBBs were also divided into four groups (G1, G3, G5 and G6) but not G2 and G4.The numbers of PtrDBB members in different groups varied; G1, G3, G5 and G6 contained four, two, two and four genes, respectively (Figure 2).To gain further understanding of the phylogenetic relationship of PtrDBB genes, a separate phylogenetic tree was constructed only using the PtrDBB protein sequences, and  the PtrDBB proteins were divided into four subfamilies, which is in accordance with the phylogenetic tree of A. thaliana, O. sativa, Z. mays, P. patens, S. moellendorffii, P. abies and A. trichopoda.To gain further information of the structural features of poplar DBB family members, the analysis of exon/intron organizations was performed by matching the PtrDBB genes' genomic DNA sequences with their cDNA sequences.Figure 3 displays the exon/intron predictions of all 12 PtrDBB genes.The number of exons in the four distant groups varied from two to six.PtrDBB10 had the highest quantities of exons (6), and PtrDBB3 and PtrDBB9 only contained two exons.However, highly similar exon/intron structures were shared in the majority of the PtrDBB genes that were assembled in the same group.For instance, two PtrDBBs contained three exons in G2 and G3, respectively, and the members within G4 had four or five exons.Overall, the phylogenetic relationship and conservated gene structure results provide a reliable basis for the classification of poplar DBB family members.

Analysis of Conservative Motif and Homology Modeling
Two putative motifs of the 12 PtrDBB proteins within each group whose lengths varied from 13 to 50 amino acids were identified using MEME 5.5.5 software, and the length of the conserved sequences and details of each motif are shown in Table S2.Comparing the MEME and conserved domain structure analysis data (Figure S1), two putative motifs were functionally annotated, which were defined as motif 1 and motif 2 that were close to

Analysis of Conservative Motif and Homology Modeling
Two putative motifs of the 12 PtrDBB proteins within each group whose lengths varied from 13 to 50 amino acids were identified using MEME 5.5.5 software, and the length of the conserved sequences and details of each motif are shown in Table S2.Comparing the MEME and conserved domain structure analysis data (Figure S1), two putative motifs were functionally annotated, which were defined as motif 1 and motif 2 that were close to N-for B-box1, motif 3 that was close to C-for B-box2, and motif 1, motif 2 and motif 3 that, together, formed the double B-box domain.However, no functional annotation was performed for the remaining five hypothetical motifs (Figure 4).The spatial distributions and motif components were varied among the different groups, but they were highly conserved within each group, which inferred that the functions of these proteins might be similar.For example, the 12 PtrDBB proteins all contained the B-box1 and B-box2 domains (motif 1, motif 2 and motif 3).The members of each subfamily not only contained the conserved double B-box domains but also had some specific motifs that may represent their diverse functions in plant development and responses to abiotic stress (Figure S1).Motif 8 (BBOX) was only presented in G3, and motif 4 (Bbox_SF superfamily) existed only in G4.Moreover, eight segmental duplications (PtrDBB1/PtrDBB11, PtrDBB1/PtrDBB3, PtrDBB2/PtrDBB6, PtrDBB5/PtrDBB7, PtrDBB8/PtrDBB12, PtrDBB9/PtrDBB11, PtrDBB9/PtrDBB12 and PtrDBB11/PtrDBB12) exhibited similar or identical motif composition structures.To obtain a better insight into the tertiary structures of the PtrDBB proteins, the protein sequences were aligned using an HMM-HMM search in intensive mode to perform the homology modeling by Phyre2 [25].The result showed that all of the 12 PtrDBB proteins could be confidently modeled and had 100% of their predicted lengths modeled, and 100% of their predicted lengths were modeled with >99% confidence (Figure 5).To obtain a better insight into the tertiary structures of the PtrDBB proteins, the protein sequences were aligned using an HMM-HMM search in intensive mode to perform the homology modeling by Phyre2 [25].The result showed that all of the 12 PtrDBB proteins could be confidently modeled and had 100% of their predicted lengths modeled, and 100% of their predicted lengths were modeled with >99% confidence (Figure 5).
To obtain a better insight into the tertiary structures of the PtrDBB proteins, the protein sequences were aligned using an HMM-HMM search in intensive mode to perform the homology modeling by Phyre2 [25].The result showed that all of the 12 PtrDBB proteins could be confidently modeled and had 100% of their predicted lengths modeled, and 100% of their predicted lengths were modeled with >99% confidence (Figure 5).

Chromosomal Location and Gene Duplications of PtrDBBs
Among the eight poplar chromosome scaffolds (Chr1, Chr2, Chr4, Chr5, Chr7, Chr9, Chr11 and Chr17) (Figure 6), chromosomes 4, 5, 7 and 9 had two PtrDBB genes, and chromosomes 1, 2, 11, and 17 possessed only one PtrDBB gene, whereas only one gene was located on the longest chromosome 1, and two genes were present on the shortest chromosome 9, which implied that the length of chromosome was not proportional to the number of genes.

Chromosomal Location and Gene Duplications of PtrDBBs
Among the eight poplar chromosome scaffolds (Chr1, Chr2, Chr4, Chr5, Chr7, Chr9, Chr11 and Chr17) (Figure 6), chromosomes 4, 5, 7 and 9 had two PtrDBB genes, and chromosomes 1, 2, 11, and 17 possessed only one PtrDBB gene, whereas only one gene was located on the longest chromosome 1, and two genes were present on the shortest chromosome 9, which implied that the length of chromosome was not proportional to the number of genes.The analysis of collinearity was conducted to elucidate the duplication events of PtrDBB homologous sequences using BLAST, taking the monocotyledons (Z.mays) and dicotyledons (A.thaliana) as controls.As a result, 31 and 19 pairs of PtrDBB genes were identified as segmental duplications between the poplar and maize genomes, and then the poplar and A. thaliana genomes, respectively (Figure 7B).Furthermore, highly conserved collinearity was found among the PtrDBB gene regions between P. trichocarpa and Z. mays, and then P. trichocarpa and A. thaliana, especially between Ptr4 and Zm5, Ptr11 and Zm1/Zm5, Ptr12 and Zm4, and then Ptr15 and Zm4, all with six synteny genes, Ptr4 and At4/At5, Ptr10/Ptr15/Ptr16 and At3/At4/At5, Ptr11 and At4/At5, Ptr12 and At3/At5, and The analysis of collinearity was conducted to elucidate the duplication events of PtrDBB homologous sequences using BLAST, taking the monocotyledons (Z.mays) and dicotyledons (A.thaliana) as controls.As a result, 31 and 19 pairs of PtrDBB genes were identified as segmental duplications between the poplar and maize genomes, and then the poplar and A. thaliana genomes, respectively (Figure 7B).Furthermore, highly conserved collinearity was found among the PtrDBB gene regions between P. trichocarpa and Z. mays, and then P. trichocarpa and A. thaliana, especially between Ptr4 and Zm5, Ptr11 and Zm1/Zm5, Ptr12 and Zm4, and then Ptr15 and Zm4, all with six synteny genes, Ptr4 and At4/At5, Ptr10/Ptr15/Ptr16 and At3/At4/At5, Ptr11 and At4/At5, Ptr12 and At3/At5, and then Ptr18/Ptr19 and At4.However, 38 duplicated pairs of PtrDBB genes were identified as segmental duplication gene pairs in a synteny map (Figure 7A

Evolution Profiles and Divergence of DBB Genes
The Ka/Ks ratios and Ks values of the 38 PtrDBB gene pairs were calculated and used to evaluate the divergence times and selective pressures of DBBs between P. trichocarpa and Z. mays, and then P. trichocarpa and A. thaliana.However, the Ka/Ks ratios of the 38 paralogous pairs (Ptr-Ptr) were the lowest at 0.103232 (Ptr2-Ptr9), followed by 0.112035 (Ptr11-Ptr12), and they were the highest at 0.378997 (Ptr11-Ptr7).In addition, the frequency distribution of the calculated average Ks values of paralogous pairs (Ptr-Ptr) was about 0.2, which suggested that the PtrDBB genes underwent a massive duplication event of approximately 15 million years ago (MYA).Compared with the previous research that suggested the genome-wide duplication of P. trichocarpa occurred 7-12 MYA, the large-scale duplication of PtrDBBs happened earlier [4].However, the frequency distributions of Ks values for the orthologous pairs from the P. trichocarpa and Z. mays, and then P. trichocarpa and A. thaliana genomes averaged ~2.43 and ~2.55 (Figure 8 and Table 3), which implied that the divergence times of the DBBs were 14 and 15 MYA, respectively.Compared with previously reported results, the divergence time between Populus and maize was 142-163, which inferred that the PtrDBB genes experienced gene evolution before the separation of these two progenitor species.The Ka/Ks ratios peaked in the poplar genome (Ptr-Ptr), and for the Ptr-Zm and Ptr-At genomes, they were distributed between 0.1 and 0.38 (Table 2) and 0.06 and 0.58 (Table 3), which suggested that the DBB genes might have gone through highly positive purifying selection.
pared with previously reported results, the divergence time between Populus and maize was 142-163, which inferred that the PtrDBB genes experienced gene evolution before the separation of these two progenitor species.The Ka/Ks ratios peaked in the poplar genome (Ptr-Ptr), and for the Ptr-Zm and Ptr-At genomes, they were distributed between 0.1 and 0.38 (Table 2) and 0.06 and 0.58 (Table 3), which suggested that the DBB genes might have gone through highly positive purifying selection.

Expression Patterns of DBB Genes in Populus trichocarpa
A hierarchical clustering heatmap was produced using the relative expression of PtrDBB genes in the different tissues of P. trichocarpa according to the published transcriptome data (Figure 9; Supplementary Materials: Table S3).This discussion showed that all 12 PtrDBB genes in the various tissues at development stages of P. trichocarpa were different.In view of the expression characteristics and hierarchical clustering of 14 poplar tissues, 12 PtrDBB genes were classified into five clusters (C1-C5).The two genes (PtrDBB11 and PtrDBB12) clustered in C1 were highly expressed in all the tissues except for Xylem1 and Phloem3.Seven genes (PtrDBB2, PtrDBB3, PtrDBB4, PtrDBB5, PtrDBB6, PtrDBB11 and PtrDBB12) grouped in C5 and C1 exhibited high expression levels in G43h, which showed that these genes are involved in the formation of G43h during poplar growth and development.Additionally, some genes exhibited similar expression patterns in different tissues; for instance, many genes (except for PtrDBB11 and PtrDBB12) exhibited high expression levels in Xylem1, and four genes (PtrDBB2, PtrDBB4, PtrDBB5 and PtrDBB6) were mainly expressed in G43h and Xylem1.Furthermore, several PtrDBB genes showed tissue-specific expression patterns, such as PtrDBB8, PtrDBB9 and PtrDBB10, which were only expressed specifically in Xylem1, while PtrDBB1 and PtrDBB7 were ubiquitously highly expressed in Xylem1 and Phloem3.Taken together, these results indicated that 12 PtrDBBs presented a variety of expression patterns in the poplar tissues at different developmental stages, which encourages future explorations of their functions and characteristics.

Cis-Regulatory Elements Analysis of PtrDBBs
The 2000 bp genomic sequence upstream from the PtrDBBs 5′-UTRs showed 41 associated cis-regulatory elements (CREs) in the promoter regions using PlantCARE (Figure 10, Additional file 5: Table S5).Among them, six CREs corresponded to light stress.These included two phytohormone-responsive elements, such as abscisic acid-(ABA) (ABRE), MeJA acid-(CGTCA-motif), SA-(TCA), IAA-(TGA and AuxRR-core) and GA-responsive elements (P-box).Detailed information of the genomic coordinates of the analyzed regions in PtrDBB genes is given in Additional file 6: Table S6.Additionally, these also included various kinds of abiotic-stress-responsive elements, including a drought-related regulatory element (MBS), a low-temperature response element (LTR) defense and stress responsiveness (TC-rich motif).It was noteworthy that each PtrDBB gene had significantly dif-

Cis-Regulatory Elements Analysis of PtrDBBs
The 2000 bp genomic sequence upstream from the PtrDBBs 5 ′ -UTRs showed 41 associated cis-regulatory elements (CREs) in the promoter regions using PlantCARE (Figure 10, Additional file 5: Table S5).Among them, six CREs corresponded to light stress.These included two phytohormone-responsive elements, such as abscisic acid-(ABA) (ABRE), MeJA acid-(CGTCA-motif), SA-(TCA), IAA-(TGA and AuxRR-core) and GA-responsive elements (P-box).Detailed information of the genomic coordinates of the analyzed regions in PtrDBB genes is given in Additional file 6: Table S6.Additionally, these also included various kinds of abiotic-stress-responsive elements, including a drought-related regulatory element (MBS), a low-temperature response element (LTR) defense and stress responsiveness (TC-rich motif).It was noteworthy that each PtrDBB gene had significantly different potential CREs, especially corresponding to light and ABA responsiveness (Figure 10; Supplementary Materials: Table S5).For example, PtrDBB1 and PtrDBB13 contained two low-temperature-responsive CREs, respectively, PtrDBB6, PtrDBB7 and PtrDBB8 contained two drought-responsive CREs, respectively.PtrDBB4, PtrDBB9, PtrDBB10, PtrDBB11 and PtrDBB12 contained two to four ABA-responsive CREs, whereas all the 12 PtrDBBs had one or more light stress elements, which suggested that most of the PtrDBBs genes took part in light regulation.

Expression Patterns of PtrDBB Genes under Abiotic Stress and Phytohormone Treatments
According to the analysis of CREs in the promoter regions, some PtrDBBs might be involved in potentially responding to different abiotic stresses and phytohormone treatments.Therefore, the expression patterns of 12 PtrDBBs under drought stress, light/dark, ABA and MeJA treatments were measured (Figure 11; Supplementary Materials: Table S7).
Under the light/dark treatments, except for the up-regulated expression levels of PtrDBB3, PtrDBB5 and PtrDBB9, the other nine genes exhibited a change in expression level of less than five-fold in comparison with that at 0 h.Among them, PtrDBB9 showed an expression level that was more than 10-fold higher during the light treatment than that during the dark treatment.
During drought stress, five PtrDBB genes (PtrDBB3, PtrDBB5, PtrDBB6, PtrDBB7 and PtrDBB9) exhibited a gradually increased expression level until 12 h and then a reduction.Furthermore, these five PtrDBB genes were the most highly expressed (>10-fold that at 0 h) after 12 h of treatment.However, the expression levels of seven genes (PtrDBB1, PtrDBB2, PtrDBB4, PtrDBB8, PtrDBB10, PtrDBB11 and PtrDBB12) changed only slightly (<5 fold that at 0 h) during the 24 h time course.
When the poplar samples were submitted to the ABA treatment, six genes (PtrDBB4, PtrDBB5, PtrDBB9, PtrDBB10, PtrDBB11 and PtrDBB12) were distinctly up-regulated.For example, five genes (PtrDBB5, PtrDBB9, PtrDBB10, PtrDBB11 and PtrDBB12) were gradually up-regulated during the early time points, peaked at 12 h, and then decreased over time.PtrDBB4 was the most highly expressed at 6 h with a gradual increase during the early time points and a significant decrease during the subsequent treatments.Moreover, PtrDBB9, PtrDBB10 and PtrDBB11 showed an expression level that was more than 10-fold higher at 12 h than that at 0 h.However, the expression levels of six genes (PtrDBB1, PtrDBB2, PtrDBB3, PtrDBB6, PtrDBB7 and PtrDBB8) changed only slightly (<5 fold than that at 0 h) during the 24 h time course.
In the MeJA treatment, four genes (PtrDBB6, PtrDBB7, PtrDBB9 and PtrDBB11) were distinctly up-regulated.For example, the expression level of two genes (PtrDBB9 and PtrDBB11) increased early, peaked at 12 h, and then decreased with time.The expression

Expression Patterns of PtrDBB Genes under Abiotic Stress and Phytohormone Treatments
According to the analysis of CREs in the promoter regions, some PtrDBBs might be involved in potentially responding to different abiotic stresses and phytohormone treatments.Therefore, the expression patterns of 12 PtrDBBs under drought stress, light/dark, ABA and MeJA treatments were measured (Figure 11; Supplementary Materials: Table S7).
Under the light/dark treatments, except for the up-regulated expression levels of PtrDBB3, PtrDBB5 and PtrDBB9, the other nine genes exhibited a change in expression level of less than five-fold in comparison with that at 0 h.Among them, PtrDBB9 showed an expression level that was more than 10-fold higher during the light treatment than that during the dark treatment.
During drought stress, five PtrDBB genes (PtrDBB3, PtrDBB5, PtrDBB6, PtrDBB7 and PtrDBB9) exhibited a gradually increased expression level until 12 h and then a reduction.Furthermore, these five PtrDBB genes were the most highly expressed (>10-fold that at 0 h) after 12 h of treatment.However, the expression levels of seven genes (PtrDBB1, PtrDBB2, PtrDBB4, PtrDBB8, PtrDBB10, PtrDBB11 and PtrDBB12) changed only slightly (<5 fold that at 0 h) during the 24 h time course.
When the poplar samples were submitted to the ABA treatment, six genes (PtrDBB4, PtrDBB5, PtrDBB9, PtrDBB10, PtrDBB11 and PtrDBB12) were distinctly up-regulated.For example, five genes (PtrDBB5, PtrDBB9, PtrDBB10, PtrDBB11 and PtrDBB12) were gradually up-regulated during the early time points, peaked at 12 h, and then decreased over time.PtrDBB4 was the most highly expressed at 6 h with a gradual increase during the early time points and a significant decrease during the subsequent treatments.Moreover, PtrDBB9, PtrDBB10 and PtrDBB11 showed an expression level that was more than 10-fold higher at 12 h than that at 0 h.However, the expression levels of six genes (PtrDBB1, PtrDBB2, PtrDBB3, PtrDBB6, PtrDBB7 and PtrDBB8) changed only slightly (<5 fold than that at 0 h) during the 24 h time course.

PtrDBBs in P. trichocarpa
The DBB transcription factor family, which is a subfamily of the B-box family, plays an essential role in regulating the circadian rhythm and early light morphogenesis.So far, the characteristic and functions of DBB genes have been determined in some plants, such as Arabidopsis [5,13], rice [9], maize [20], pepper [26,27], cotton [28], tomato [29], apple [30], berry [31] and so on [32].Six, eight, ten and nine more poplar DBB subfamily members were found compared to those in Physcomitrella patens (six), Selaginella moellendorffii (four), Picea abies (two) and Amborella trichopoda (three), respectively.Additionally, the motifs and gene structures in the same subfamily were varied among different branches, but they were highly similar in the same phylogenetic branch.For example, in subfamily I, PtrDBB2 and PtrDBB6 had the same motifs (motifs 1, 2, 3, 4, 5, 6 and 7), and PtrDBB9 and PtrDBB3 contained all the same motif structures (motif 1, 2, 3 and 5).It was determined that PtrDBBs in the same phylogenetic branch have a similar motif structure, leading to a consistent evolutionary pattern of PtrDBB transcription factors.Moreover, the intron/exon In the MeJA treatment, four genes (PtrDBB6, PtrDBB7, PtrDBB9 and PtrDBB11) were distinctly up-regulated.For example, the expression level of two genes (PtrDBB9 and PtrDBB11) increased early, peaked at 12 h, and then decreased with time.The expression of one gene, PtrDBB6, was up-regulated significantly, reaching a value that was approximately 13-fold higher at 6 h, while the expression of eight genes (PtrDBB1, PtrDBB2, PtrDBB3, PtrDBB4, PtrDBB5, PtrDBB8, PtrDBB10 and PtrDBB12) presented insignificant changes (a change in expression levels of less than five-fold at all times).
Overall, PtrDBB5 and PtrDBB9 were the only two genes which were significantly up-regulated in response to all the abiotic stress and different phytohormone treatments.

PtrDBBs in P. trichocarpa
The DBB transcription factor family, which is a subfamily of the B-box family, plays an essential role in regulating the circadian rhythm and early light morphogenesis.So far, the characteristic and functions of DBB genes have been determined in some plants, such as Arabidopsis [5,13], rice [9], maize [20], pepper [26,27], cotton [28], tomato [29], apple [30], berry [31] and so on [32].Six, eight, ten and nine more poplar DBB subfamily members were found compared to those in Physcomitrella patens (six), Selaginella moellendorffii (four), Picea abies (two) and Amborella trichopoda (three), respectively.Additionally, the motifs and gene structures in the same subfamily were varied among different branches, but they were highly similar in the same phylogenetic branch.For example, in subfamily I, PtrDBB2 and PtrDBB6 had the same motifs (motifs 1, 2, 3, 4, 5, 6 and 7), and PtrDBB9 and PtrDBB3 contained all the same motif structures (motif 1, 2, 3 and 5).It was determined that PtrDBBs in the same phylogenetic branch have a similar motif structure, leading to a consistent evolutionary pattern of PtrDBB transcription factors.Moreover, the intron/exon structure of PtrDBBs in the same subfamily had some differences, but this was highly similar within the same phylogenetic branch.Thus, the conservation and diversity of motif and intron/exon structures are of great significance for studying the evolution of gene families.

Evolutionary Patterns of DBBs in P. trichocarpa
In view of the phylogenetic relationship analysis data, the poplar DBB proteins were closely related to Arabidopsis DBB proteins, which are also a dicotyledon, and among them, PtrDBB1/PtrDBB11 and AtDBB3, PtrDBB12/PtrDBB6 and AtSTH2, PtrDBB3/PtrDBB9 and AtDBB6, and PtrDBB5/PtrDBB7 and AtDBB1a/AtDBB1b showed more similarity.Furthermore, this genetic relationship is followed by O. sativa and Z. mays, and PtrDBB4/PtrDBB10 was very similar to OsDBB1 in O. sativa and ZmDBB10 in Z. mays.In addition, the poplar DBB family was very different from Selaginella moellendorffii, Picea abies and Amborella trichopoda.
Gene duplication is a crucial method of gene family amplification in the genomic evolution of plants [33]; moreover, this helps organisms adapt to various environments during their development [6].In the poplar DBB gene family, 20 segmental gene pairs and no tandem gene pairs were identified, suggesting that gene segmental duplication was the major driving force for the expansion of the DBB gene family in P. trichocarpa.The chromosomal localization of the PtrDBB gene suggests that the distribution of the DBB gene in the poplar genome may be the result of genome replication events [34].Six pairs of potential duplicate DBB genes appeared in different poplar chromosomes.In addition, the collinearity analysis of Populus and maize genome sequences identified 21 PtrBBX-ZmBBX gene pairs and showed that there was a significant collinearity between P. trichocarpa and monocots Z. mays, which was consistent with the evolutionary relationship between the dicotyledons and monocotyledons.

Potential Roles of PtrDBBs in P. trichocarpa
DBBs participate in plant growth and development, such as in seedling photomorphogenesis, flowering time, phytochromes, pigmentation and cotyledon development in Arabidopsis [5,13], rice [9], maize [20], pepper [26,27], cotton [28] and tomato [29].In this study, the expression levels of 12 PtrDBBs were examined in 14 tissues at different development stages on the basis of previously reported transcriptome data (Supplementary Materials: Table S3).In view of the results, the PtrDBB genes in these different tissues might take part in the control of plant growth and development, and some PtrDBBs might have particular functions in specific tissues and at different developmental stages.For example, all of the PtrDBBs (except for PtrDBB11 and PtrDBB12) showed relatively high expression levels in xylem1, implying their potential role in xylem1 development.PtrDBB11, an ortholog of AtDBB3, was highly expressed in most of the tissues except for phloem3 and xylem1.Likewise, PtrDBB12 also showed a similar expression pattern in these tissues, indicating their essential functions in P. trichocarpa.Moreover, PtrDBB2, PtrDBB3 and PtrDBB6 were the most homologous to AtSTH2, and the expression levels in G43h and xylem1 were significantly higher than those in the other tissues.AtSTH2 is a regulator of light signal transduction, indicating that its homologous genes PtrDBB2, PtrDBB3 and PtrDBB6 might be involved in early photomorphogenesis [35].Other researchers have found that AtSTH2 is able to communicate with HY5 in both yeast and plants, indicating that some DBB proteins, along with HY5 (LONG HYPOOTYL 5), are involved in the complicated early photomorphogenesis of higher plants [14].In Arabidopsis, HY5 positively regulates light signal transduction but negatively regulates photomorphogenesis without light [13].Therefore, PtrDBB2, PtrDBB3 and PtrDBB6 might have similar light signal transduction functions in P. trichocarpa.

Stress and Phytohormones Induced Expression of PtrDBBs in P. trichocarpa
Many abiotic stresses, such as cold and high temperatures, drought, waterlogging, salinity, metals and nutritional deficiencies can cause a variety of stress response mechanisms, afterwards activating the correlation genes required for stress tolerance [36].A large number of stress response CREs (41) were present in the promoter regions of the 12 PtrDBB genes, indicating their potential roles in stress response.For example, all the 12 genes contained the light-responsive CREs in their promoters, ranging from one to seven.Li et al. [20] has found that ZmDBB5, ZmDBB9 and ZmDBB10 are up-regulated when poplar is treated with a light/dark treatment.Similar expression patterns of PtrDBB3 and PtrDBB9 (homologous of ZmDBB5 and ZmDBB9) and PtrDBB5 (homologous of ZmDBB10) were identified in this study, implying their vital functions in response to a light/dark treatment.
Phytohormones act as important regulators in normal plant growth and development, and auxin, gibberellin, cytokinin, abscisic acid and ethylene are the major phytohormones.The gene family members in Arabidopsis and other plant species can respond to different phytohormones [37].The previous studies showed that DBB1a acted as a negative regulator of circadian rhythm and light signals and participates in the gibberellin homeostasis [13].In addition, six DBB genes in maize exhibited differential expression patterns under various phytohormone treatments, indicating that these ZmDBB genes may take part in different phytohormone signaling pathways [20].In this study, 12 PtrDBB genes presented differential expression patterns when challenged with ABA and MeJA treatments, and the majority of them contained CREs that respond to phytohormones in their promoter regions.The expression levels of three genes (PtrDBB5, PtrDBB9 and PtrDBB11) were significantly increased under the ABA and MeJA treatments, and all of them harbored ABRE and CGTCA in their promoter regions.Similarly, PtrDBB3, PtrDBB5 and PtrDBB9 were induced by the light/dark treatment and drought stress, and they all contained 2-10 light-responsive CREs in their promoter regions.In addition, two genes (PtrDBB5 and PtrDBB9) were discovered to respond to the light, drought stress, ABA and MeJA treatments, indicating their potential roles in a cross connection between the phytohormones and light signals.Therefore, it has been shown that these PtrDBB genes may act as transcriptional regulators to participate in phytohormone signaling pathways and to regulate the plants' responses to various abiotic stresses.

Plant Material and Growth Conditions
One-hundred-day-old poplar 84 K (Populus alba × Populus glandulosa) trees grown in a standard greenhouse (light/dark cycle: 16 h/8 h at 25 • C; 85% relative humidity) at the Beijing Forestry University poplar nursery planting base (Beijing, China) were selected as the experimental material.For drought stress treatment, the seedlings were treated with polyethylene glycol (PEG) 6000 (25%).Moreover, the seedlings were treated with MS medium solution (100 mmol/L), 200 µM abscisic acid (ABA), or 200 µM methyl jasmonate (MeJA), respectively [29,38].The light/dark treatment and daytime treatments were conducted as mentioned by Li et al. [20].The seedlings were sprayed with Murashige and Skoog (MS) liquid medium supplemented with 200 µM ABA or MeJA.The control seedlings were only sprinkled with MS medium solution.

Chromosomal Location, Synteny Analysis and Duplication Events
The detailed chromosomal locations of PtrDBB genes were retrieved from the Gene Structure Shower of TBtools [48] by importing the genome annotation files and gene IDs and visualizing them using Circos [49].MapInspect 1.0 software was used to map the physical location of PtrDBBs on the corresponding poplar chromosomes (https://mybiosoftware. com/mapinspect-compare-display-linkage-maps.html, 1 January 2023).Possible genome replication gene pairs of P. trichocarpa, Z. mays and A. thaliana, and duplication events were detected using MCScanX-2019 software (http://chibba.pgml.uga.edu/mcscan2/#tm, 1 January 2023).MCScanX software was also used to perform the collinearity analysis of GLK proteins with BLAST comparing the sequences [50].

PtrDBB Expression Profiles and qRT-PCR Analysis
A heatmap was created using TBtools to exhibit the expression patterns of PtrDBBs.RNAseq data of PtrDBB genes in 14 tissues were collected according to Rodgers-Melnick et al. [51].The NCBI Primer-BLAST tool was used to design the primers of the 12 PtrDBB genes, which could amplify the 100-200 bp PCR products (Additional file: Table S4).Total RNA was isolated from the samples of each tissue using an RNAprep Pure Plant Kit (TransGen Biotech, Beijing, China) according to the user manual.Total RNAs were used for complementary cDNA synthesis using SuperScript III transcriptase (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions.qRT-PCR analysis was performed on a Bio-Rad CFX96 using the Light Cycler 480 SYBR Green Master Mix (TaKaRa, Dalian, China).The PCR reaction conditions were as follows: 95 • C for 30 s, followed by 40 cycles of 95 • C for 5 s, and 60 • C for 30 s.The quantitative RT-PCR data were analyzed using the 2 −∆∆Ct method.The mean expression values and SE values were calculated from the results of three independent experiments.

Conclusions
In this study, 12 poplar DBB genes were identified and divided into four subfamilies based on their gene and motif structures, conserved domains and phylogenetic relationships.In addition, the bioinformatic analysis of chromosome location, collinearity and gene duplication has aided our understanding of the biological functions of poplar DBB genes.Several PtrDBB genes expressed tissue specificity and diversity at various stages, and some were involved in the abiotic stress and phytohormone treatment responses.The analysis of transcriptomes and qRT-PCR data suggested that PtrDBBs might contribute to poplar development by regulating multiple phytohormone signaling pathways.Overall, the genome-wide analysis of DBBs is the basis for the potential function analysis of PtrDBBs in P. trichocarpa, and further research is underway on several DBBs to explore their biological functions.

Figure 3 .
Figure 3. Analysis of phylogenetic relationship and DBB gene structures by MEGA-X in view of the PtrDBB protein sequences in P. trichocarp.Left: a neighbor-joining (NJ) phylogenetic tree was constructed.All the PtrDBB genes were divided into four clades, and different groups were represented by different colors.Right: the analysis of exon/intron structures of 12 PtrDBB genes.The yellow and green rectangles represent exons and introns, respectively.The black lines represents untranslated regions (UTRs).

Figure 3 .
Figure 3. Analysis of phylogenetic relationship and DBB gene structures by MEGA-X in view of the PtrDBB protein sequences in P. trichocarp.Left: a neighbor-joining (NJ) phylogenetic tree was constructed.All the PtrDBB genes were divided into four clades, and different groups were represented by different colors.Right: the analysis of exon/intron structures of 12 PtrDBB genes.The yellow and green rectangles represent exons and introns, respectively.The black lines represents untranslated regions (UTRs).
and PtrDBB11/PtrDBB12) exhibited similar or identical motif composition structures.

Figure 4 .
Figure 4. Distribution of conserved motifs for PtrDBB proteins (1-8).The analysis of PtrDBBs conserved motifs was carried out by the MEME.Eight motifs were displayed by boxes of different colors, and the lengths of the motif were represented in proportion.The annotation information of each motif is marked in the bottom right corner.

Figure 4 .
Figure 4. Distribution of conserved motifs for PtrDBB proteins (1-8).The analysis of PtrDBBs conserved motifs was carried out by the MEME.Eight motifs were displayed by boxes of different colors, and the lengths of the motif were represented in proportion.The annotation information of each motif is marked in the bottom right corner.

Figure 6 .
Figure 6.Chromosomal distribution of poplar DBB genes.Different colors indicated the grouping of PtrDBB genes.Six segmental duplication pairs were connected by orange lines.

Figure 6 .
Figure 6.Chromosomal distribution of poplar DBB genes.Different colors indicated the grouping of PtrDBB genes.Six segmental duplication pairs were connected by orange lines.

Figure 7 .
Figure 7. Duplication events of DBB genes.(A) Synteny of poplar DBB genes.(B) Synteny of P. trichocarpa and Z. mays, P. trichocarpa and A. thaliana DBB gene regions.Segmental duplicated DBB gene pairs, and duplicated blocks were linked by red lines and gray lines, respectively.

Figure 8 .
Figure 8. Ks and Ka/Ks value distribution of the DBB genes in the genomes of poplar paralogous gene pairs (Ptr-Ptr) and orthologous gene pairs between P. trichocarpa and Z. mays (Ptr-Zm), P. trichocarpa and A. thaliana (Ptr-At), viewed from the frequency distribution.

Figure 8 .
Figure 8. Ks and Ka/Ks value distribution of the DBB genes in the genomes of poplar paralogous gene pairs (Ptr-Ptr) and orthologous gene pairs between P. trichocarpa and Z. mays (Ptr-Zm), P. trichocarpa and A. thaliana (Ptr-At), viewed from the frequency distribution.

Molecules 2024 ,
29, x FOR PEER REVIEW 11 of 19highly expressed in Xylem1 and Phloem3.Taken together, these results indicated that 12 PtrDBBs presented a variety of expression patterns in the poplar tissues at different developmental stages, which encourages future explorations of their functions and characteristics.

Figure 10 .
Figure 10.Analysis of cis-elements of PtrDBBs using the Plantcare database.

Molecules 2024 , 19 Figure 11 .
Figure 11.The expression of 12 PtrDBB genes under different stresses (light/dark, drought, ABA, and MeJA treatments).Compared with untreated samples (expression levels = 1) after sampling to analyze the relative expression levels.X-axes and Y-axes mean time points after light/dark stress, drought stress, ABA, and MeJA treatments, and data were normalized to reference gene Ptr18S, respectively.Three independent biological replicates were conducted.

Figure 11 .
Figure 11.The expression of 12 PtrDBB genes under different stresses (light/dark, drought, ABA, and MeJA treatments).Compared with untreated samples (expression levels = 1) after sampling to analyze the relative expression levels.X-axes and Y-axes mean time points after light/dark stress, drought stress, ABA, and MeJA treatments, and data were normalized to reference gene Ptr18S, respectively.Three independent biological replicates were conducted. .
and Table2), which implied that the amplification of the poplar DBB gene family depends on segmental replication events.

Table 3 .
Ka/Ks of orthologous GLK genes pairs (Ptr-At, Ptr-Zm) in P. trichocarpa and two species (A.thaliana and Z. mays).a million years ago.

Table 3 .
Ka /Ks of orthologous GLK genes pairs (Ptr-At, Ptr-Zm) in P. trichocarpa and two species (A.thaliana and Z. mays).a million years ago.