PyunBBX18 Is Involved in the Regulation of Anthocyanins Biosynthesis under UV-B Stress

(1) Background: Populus yunnanensis Dode (P. yunnanensis) grows in the low-latitude and high-altitude areas of southwest China. In low-latitude and high-altitude areas, plants suffer from the high intensity of UV-B (ultraviolet-b) radiation, and they have a complete regulation system to adapt to the environment of the high UV-B radiation. As natural antioxidants, anthocyanins play an important role in scavenging free radicals. BBX (B-box) genes are involved in anthocyanins biosynthesis. (2) Methods: By exploring the gene structure and motifs of PyunBBX genes (genes of P. yunnanensis BBX family) and the evolutionary relationship between PyunBBX genes and other species BBX genes, six PyunBBX genes that responded to UV-B and participated in anthocyanins biosynthesis were screened. BBX, with the potential to regulate anthocyanins biosynthesis, was further investigated by anthocyanins content determination and RT-qPCR (real-time quantitative polymerase chain reaction); (3) Results: After 7 days of UV-B treatment, anthocyanins were significantly accumulated, and the expression of PyunBBX18 was up-regulated for 7 days. The expression of PyunBBX12 was inhibited by UV-B treatment. By analyzing the RNA-seq data of leaves and bark of P. yunnanensis, we found that PyunBBX18 was highly expressed in leaves and young bark; (4) Conclusions: These results showed that PyunBBX18 and PyunBBX12 may be involved in the response process of UV-B stress, in which PyunBBX18 may regulate the anthocyanins biosynthesis to resist UV damage.


Introduction
Transcription factors (TFs) play an important role in plant growth and development and stress defense. BBX is a kind of TF containing a zinc finger structure that mediates and regulates many biological processes in plants, including photomorphogenesis, flowering regulation, and response to biological and abiotic stresses [1]. The N-terminal of the BBX protein contains one B-box-conserved domain or two B-box-conserved domains, namely B-box1 and B-box2. Some BBX have a CCT (CONSTANS, CO-like, and TOC1) domain at the C-terminal. The B-box conserved domain is involved in protein interaction and transcriptional regulation, while the CCT domain is mainly involved in nuclear transport and transcriptional regulation [2,3].
Over the years, research has shown that the majority of BBX gene family members can interact with HY5 (ELONGATED HYPOCOTYL5, an important TF of light signal) or regulate transcription of HY5, thereby regulating photomorphogenesis and anthocyanins biosynthesis in plants [4][5][6][7][8][9][10]. For instance, AtBBX21 binds to T/G-box in the AtHY5 promoter through its second B-box domain, thereby regulating the expression of AtHY5 and HY5-regulated genes to promote photomorphogenesis in plants [5]. Under UV-B treatment, the module SIBBX20/21-SIHY5 could activate the transcription of SIHY5, and the excessive

Identification of BBX Gene Family Members in P. yunnanensis
The genome at the chromosome level of P. yunnanensis was obtained from our laboratory group (Southwest Forestry University, Kunming 6500224, China). In order to identify the BBX in P. yunnanensis genome, firstly, the 32 members of BBX proteins sequences in Arabidopsis thaliana (A. thaliana) genome were downloaded from the TAIR 10 (https://www.arabidopsis.org/, accessed on 1 December 2021) database [3]. The 32 proteins sequences were used to search the BBX family members in P. yunnanensis by Blastp (E-value < 1E-05) [31]. The profile of the hidden Markov model (HMM) for the B-box domain (Pfam00643) and CCT domain (Pfam06203) was obtained from Pfam (https://pfam. xfam.org/, accessed on 2 December 2021) [32]. The hmmsearch program of the HMMER 3.0 software was used to search for the putative BBX gene family members in the P. yunnanensis genome, and the E-value was set to 1E-05 [33]. If a gene had multiple transcripts, the longest transcript was selected as the representative protein sequence for further analysis. After removing the duplicate for candidate BBX gene family members, the reserved protein sequences of BBX gene family members were submitted to the SMART database (http://smart.embl-heidelberg.de/, accessed on 3 December 2021) [34], CDD (Conserved Domain Database, https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml, accessed on 3 December 2021) [35], and InterPro database (http://www.ebi.ac.uk/interpro/, accessed on 3 December 2021) [36] in order to analyze whether it contained unique B-box conservative domain structure. The B-box conservative domain structure of candidate genes was identified as the BBX gene family members of P. yunnanensis.

Gene Structure and Motif Analysis of PyunBBX Genes
The length and location of exons and introns of PyunBBX genes were obtained from the P. yunnanensis genome for further analysis. Each sequence of PyunBBX genes was submitted to MEME (https://meme-suite.org/meme/tools/meme, accessed on 18 December 2021) [39]. In the conservative base sequence analysis, the maximum was set to 10, the mode was selected as zoops, and the default values of other parameters were used. Finally, the results were visualized by TBtools [40] and WebLogo (http://weblogo.berkeley.edu/logo.cgi, accessed on 20 December 2021).

Cis-Acting Elements Analysis
The promoter-upstream region (~2000 bp) sequences were extracted from the genomic DNA sequence of P. yunnanensis, and the sequences were submitted to the Plant-CARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 2 January 2022) [41] for cis-acting element prediction. Excel was used to sort out and simplify the analysis results, and the cis-acting elements with the same biological functions were labeled with the same label notes.

Chromosomal Location, Gene Duplication, and Synteny Analysis
The chromosome location image of the PyunBBX genes was drawn using the TBtools software according to the physical positions of PyunBBX genes on the P. yunnanensis chromosomes. All the BBX amino acid sequences of A. thaliana, P. trichocarpa, and P. yunnanensis were included in a local database using DIAMOND [42]. The P. trichocarpa genome (version = 3.0) was derived from the Phytozome database (https://phytozome-next.jgi. doe.gov/info/Ptrichocarpa_v3_0, accessed on 15 March 2022). The BBX protein sequences of P. yunnanensis were used as queries to search the above-mentioned database with an E-value of 1E-05. The blast results were analyzed by the MCScanX (Multiple Collinear Scan Kit) [43] and TBtools to determine and analyze the duplication and synteny of BBX genes. The Ks (synonymous substitution rate) and Ka (nonsynonymous substitution rate) values of collinearity pairs in PyunBBX genes were estimated by the KaKs_calculator [44] procedure in TBtools. The divergence time was calculated with the formula T = Ks/2r, with Ks being the synonymous substitutions per site and r being the rate of divergence for nuclear genes from plants. The r was taken to be 1.5 × 10 −8 synonymous substitutions per site per year for dicotyledonous plants [45].

Evolutionary Analysis of BBX Gene Family Members
The molecular evolutionary genetic analysis tool MEGA (Molecular Evolutionary Genetics Analysis) version 7.0.20 [46] was used for multiple sequence alignment and evolutionary analysis. The ClustalW program of MEGA was firstly used for multi-sequence alignment of the BBX amino acid sequences. Then, the gaps were manually sheared, and conservative region sequences were reserved. The maximum likelihood (ML) method was selected to construct the phylogenetic tree for PyunBBX genes, and the neighbor-joining (NJ) method constructed a phylogenetic tree for BBX genes of multispecies. Bootstrap was set to 1000.

Plant Materials, Growth Conditions, and UV Treatment
The plant material was from the branches of P. yunnanensis that had grown for one year in the greenhouse of Southwest Forestry University. They were pruned into cuttings of about 15 cm, and cuttings were made in March 2022. The cuttings were induced when they had reached 5-6 leaves. The cuttings of the same growth were placed in the constant temperature incubator (temperature: 25°C, humidity: 70%, light intensity: 90 µmol·m −2 ·s −1 , photoperiod: 12 h light/12 h dark) for cultivation, a UV lamp (Telipu, Beijing Zhongyi Boteng Technology Company, power: 8 W, radiation intensity: 68 µW/cm 2 , wavelength: 280-320 nm) was placed 35 ± 1 cm above the cuttings, and UV irradiation was carried out for 12 h every day under normal light treatment. The cottage seedlings were sampled after UV treatment for 12 h, and the sampling time was 0, 1, 2, 3, 5, and 7 days. The control group was cottage seedlings that grew under normal light. The sampling sites were the second and third fully extended leaves. The samples were frozen with liquid nitrogen and immediately stored in the −80 • C refrigerator.

Total RNA Isolation and cDNA Synthesis
Total RNA was isolated from the leaf of P. yunnanensis using the E.Z.N.A. @ Plant RNA kit (Omega Bio-tek Inc., Norcross, USA) according to its manual. The quality of RNA was evaluated with K5800C (KAIAO, Beijing, China). A total of 500 ng RNA of each sample was used for 1st strand cDNA synthesis using Hifair ® III Reverse Transcriptase (YEASEN, Shanghai, China) according to the manufacturer's protocols. cDNA was diluted 8-fold for RT-qPCR analysis.

Quantitative Real-Time PCR
Primers were designed based on CDS sequences for real-time PCR by using NCBI primer-blast (Table S1). The internal reference gene was HIS (histone) [47]. Real-time PCR application was carried out in a LightCycler ® 96 Real-Time PCR Detection System (Roche, Hercules, Switzerland) with Hieff UNICON ® Universal Blue qPCR SYBR Green Master Mix (YEASEN, Shanghai, China). A total of 20 µL reaction system contained 10 µL Blue qPCR SYBR Green Master Mix, 1 µL cDNA samples, 0.4 µL of each primer (1 µM), and 8.2 µL ddH2O. The PCR thermal cycle conditions were as follows: denaturation at 95 • C for 2 min, 45 cycles of 95 • C for 10 s, and 56 • C for 30 s. Fluorescence intensities were measured for RT-qPCR at the end of each cycle. A melting curve (1 cycle of 95 • C for 10 s, 65 • C for 10 s, and 97 • C for 1 s) was performed directly to check for specific amplification. The relative gene expression was calculated by using the 2 − Ct method [48], and the experiments were performed in triplicate technological repeats.

Extraction and Determination of Anthocyanins
Determination of anthocyanins content used ethanol/hydrochloric acid assay [49]. Fresh leaves were cut into pieces and weighed with 0.1 g, placed in a 10 mL centrifuge tube, and 10 mL hydrochloric acid ethanol (0.1 mol/L) solution was added to the tube. It was warmed at 60 • C for 30 min in a water bath. Then, the supernatant was transferred to a 25 mL volumetric flask after cooling. Then, 5 mL hydrochloric acid ethanol solution was added to the tube and continued water bath for 15 min. This step was repeated three times, and the supernatant was combined. Finally, the volume was fixed to 25 mL, and the absorbance value (Abs) was measured at 530 nm. Substitute the measured results into the following formula for calculation: Genes 2022, 13, 1811 5 of 21 C: anthocyanins content (mg/100 g), A: Abs (nm), V: metered volume (mL), m: mass (g), 958: empirical coefficient of Abs conversion into mass volume ratio, 10 3 : unit conversion coefficient.

Expression Patterns of BBX Family Members in P. yunnanensis by Rna-seq Data
Transcriptome data of leaves (PRJNA505895) and bark (PRJNA542544) at different mature sites (PRJNA506110) were downloaded from NCBI (National Center for Biotechnology Information). For raw data, fastQC (Version = 0.11.9) was used for quality inspection. Fastp (Version = 0.19.4) further filtered low-quality reads. The obtained high-quality clean reads were compared to the genome of P. yunnanensis using Hisat2 (version = 7.5.0). FeaturCounts (Version = 2.0.1) normalized FPKM (fragments per kilobase of exon model per million mapped fragments) values were obtained in the R language for expression pattern analysis. Heatmap (Version = 2.8.0) was used to draw the heat map of gene expression.

Data Analysis
Adobe Photoshop CS6 was used for image processing, and Adobe Illustrator 2020 was used for chart layout. The data obtained from the experiment were plotted and analyzed by Graphpad Prism 8.0.2 software.

Analysis of The Basic Characteristics of PyunBBX Genes
Totally, 43 PyunBBX genes were identified from the P. yunnanensis genome. They were named PyunBBX1 to PyunBBX43 in the order of their position on chromosomes. They had different sequence lengths, resulting in different isoelectric points and molecular weights ( Table 1). Amino acid sequences ranged in length from 184 aa (PyunBBX10) to 513 aa (PyunBBX1). The predicted molecular weight of the BBX gene was 12.11 kDa (PyunBBX5) to 59.46 kDa (PyunBBX9), and the theoretical isoelectric point was 4.12 (PyunBBX30) to 9.26 (PyunBBX29). Subcellular prediction showed that all of the PyunBBX genes were located in the nucleus. On 19 chromosomes of the P. yunnanensis genome, there were no PyunBBX genes on chromosomes 12, 13, and 19, and the other chromosomes (chromosomes 1-11 and chromosomes 14-18) had PyunBBX genes ( Figure 1).

Gene Structure and Characteristics of Conserved Sequences of PyunBBX Gene Family Members
The amino acid sequences of 43 members of the P. yunnanensis BBX gene family were analyzed, and the 3 most significant conserved sequences were extracted, namely B-box1, B-box2, and CCT (Figures 2, 3 and S1). B-box1 and B-box2 have a high similarity, with the structure of C 2 -X 2 -C-X 7, 8 -C-X 7 -C-X 2 -C-D-X n -H. Besides PyunBBX5, each of them has five conserved cysteine (Cys, C) residues adjacent to aspartic acid (Asp, D). Moreover, the Cys on both sides are (Leu, L) linked. Such a structure can combine with Zn 2+ and form a "finger" structure through self-folding. It can be further combined with RNA, DNA, and protein to carry out gene regulation at the level of transcription and translation. Some BBX members also have a CCT domain, which is R- containing a large number of arginine (Arg, R), followed by conservative tyrosine (Tyr, Y) and lysine (Lys, K), participating in the nuclear localization of the protein [50].
The structure of PyunBBX genes is shown in Figure 3. The results showed that most PyunBBX genes contained at least one intron and up to five introns. PyunBBX genes could be divided into five groups. It was consistent with the classification of other species. The first group contains 12 PyunBBX genes with 2 B-box domains and 1 CCT domain. The second group contains six PyunBBX genes with two B-box domains and one CCT domain. The third group contains three PyunBBX genes with a B-box domain and a CCT structure. The fourth group contains 14 members of PyunBBX genes with 2 B-box domains. The fifth group contains eight PyunBBX genes with a B-box domain. In addition, some PyunBBX genes (PyunBBX12/17/18/21/37/38/41/42/43) also have a VP motif, and the VP peptide motif in the gene can bind to the WD40 domain of COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) and interact with it to participate in the light response [51].

Gene Structure and Characteristics of Conserved Sequences of PyunBBX Gene Family Members
The amino acid sequences of 43 members of the P. yunnanensis BBX gene family were analyzed, and the 3 most significant conserved sequences were extracted, namely B-box1, B-box2, and CCT (Error! Reference source not found. Figure 2, 3, and S1). B-box1 and B- structure can combine with Zn 2+ and form a "finger" structure through self-folding. It can be further combined with RNA, DNA, and protein to carry out gene regulation at the level of transcription and translation. Some BBX members also have a CCT domain, which is R-X5-R-Y-X-E-X4-R-X3-K-X2-R-Y-X3-K-X5-R-X-R-X2-G, containing a large number of arginine (Arg, R), followed by conservative tyrosine (Tyr, Y) and lysine (Lys, K), participating in the nuclear localization of the protein [50].

Collinearity Analysis
The high sequence similarity between repetitive gene pairs indicated that they were likely involved in regulating similar biological processes. MCScanX was used to analyze the collinear blocks and gene duplication type. There were 14 gene pairs of PyunBBX genes, which were PyunBBX1/9, PyunBBX6/16, PyunBBX15/19, PyunBBX13/26, PyunBBX14/27, PyunBBX18/42, PyunBBX23/28, PyunBBX22/29, PyunBBX4/31, PyunBBX34/35, PyunBBX21/37, PyunBBX20/39, PyunBBX12/41, and PyunBBX17/43, and they belong to segmental in duplicated type (Figure 4, Table 2). The Ka/Ks values of 14 pairs of collinear blocks were all less than 1, indicating that these members may have been affected by purification selection in evolution. By calculating the divergence time of collinear blocks, the result showed that the average differentiation time of PyunBBX genes was 2.3 Mya (millions of years ago). Moreover, PyunBBX22 and PyunBBX29 differentiated at 3.92 Mya that they had the longest time of differentiation, and PyunBBX34 and PyunBBX35 differentiated at 0.24 Mya that they diverged more recently (Table 2). We constructed comparative syntenic maps between P. yunnanensis and the other two species (P. trichocarpa and A. thaliana) to analyze the similarities of the BBX gene among species. Syntenic maps revealed that 86 pairs of homologous genes were found between P. trichocarpa and P. yunnanensis, and 46 pairs of homologous genes were found between P. yunnanensis and A. thaliana ( Figure 5). The structure of PyunBBX genes is shown in Figure 3. The results showed that most PyunBBX genes contained at least one intron and up to five introns. PyunBBX genes could be divided into five groups. It was consistent with the classification of other species. The

Collinearity Analysis
The high sequence similarity between repetitive gene pairs indicated that they were likely involved in regulating similar biological processes. MCScanX was used to analyze the collinear blocks and gene duplication type. There were 14 gene pairs of PyunBBX genes, which were  (Figure 4, Table 2). The Ka/Ks values of 14 pairs of collinear blocks were all less than 1, indicating that these members may have been affected by purification selection in evolution. By calculating the divergence time of collinear blocks, the result showed that the average differentiation time of PyunBBX genes was 2.3 Mya (millions of years ago). Moreover, PyunBBX22 and PyunBBX29 differentiated at 3.92 Mya that they had the longest time of differentiation, and PyunBBX34 and PyunBBX35 differentiated at 0.24 Mya that they diverged more recently ( Table 2).  We constructed comparative syntenic maps between P. yunnanensis and the other two species (P. trichocarpa and A. thaliana) to analyze the similarities of the BBX gene among species. Syntenic maps revealed that 86 pairs of homologous genes were found between P. trichocarpa and P. yunnanensis, and 46 pairs of homologous genes were found between P. yunnanensis and A. thaliana ( Figure 5).

Cis-Acting Elements of PyunBBX Genes Analysis
The promoter-upstream region (~2000 bp) sequences were extracted from the genomic DNA sequence of PyunBBX genes. The cis-elements of the PyunBBX gene family promoters were analyzed by using the PlantCARE database ( Figure 6, Table S2). Each promoter of PyunBBX genes contains abiotic and biotic stress response elements (such as Figure 5. Synteny analysis between P. yunnanensis and the other two species. The putative collinear genes between P. yunnanensis and the other two species are marked in gray, while the syntenic BBX gene pairs are marked in red.

Expression Patterns of PyunBBX Genes in Leaf and Bark of P. yunnanensis by Rna-seq Data
The expression patterns of PyunBBX genes in leaf and bark were analyzed using RNAseq data of leaves and bark in NCBI. Compared to the bark, PyunBBX12/15/18/29/36/37/38 expression quantity is relatively high in the leaves. The expression level of PyunBBX13 was very low in bark, with almost no expression (average of FPKM= 0.014062), while it was expressed in leaves (average of FPKM = 4.59). Compared with the leaves, PyunBBX3/4/6/8/14/ 16/27/28/31/32/40 expressed in the bark of the organization's quantity is relatively high (Figure 7). The expression patterns of PyunBBXs in bark tissues at different locations showed that the expression patterns of most PyunBBXs were basically consistent, indicating that they performed the same function in bark tissues at different locations ( Figure S2) [52]. The expression level of PyunBBX16/36/42 in the younger bark position (EU, Figure S2) was lower than that of other bark tissues, and the expression level of PyunBBX18 in EU was higher than that of other bark tissues (Figure 8).
phytohormone correlation (such as as-1, TCA-element, W box, CGTCA-motif, TGACGmotif, ABRE, AAGAA-motif, ERE, p-box, GARE-motif, TATC-box, AuxRR-core, and TGA-element), and light responsiveness (such as G-Box, MRE, GT1-motif, Sp1, ACE, 3-AF1 binding site, and AAAC-motif). Some PyunBBX genes had the cis-acting elements of the MYB binding site involved in light responsiveness, flavonoid biosynthetic genes regulation, and drought-inducibility (Table S2). This showed the functional diversity of the PyunBBX genes.  organization's quantity is relatively high (Figure 7). The expression patterns of PyunBBXs in bark tissues at different locations showed that the expression patterns of most PyunBBXs were basically consistent, indicating that they performed the same function in bark tissues at different locations ( Figure S2) [52]. The expression level of PyunBBX16/36/42 in the younger bark position (EU, Figure S2) was lower than that of other bark tissues, and the expression level of PyunBBX18 in EU was higher than that of other bark tissues (Figure 8).

Expression Patterns of Six PyunBBXs under UV Treatment by RT-qPCR Analysis
In order to determine the PyunBBX genes in response to UV, we picked out the highly expressed PtrBBXs of P. trichocarpa under the UV-B treatment (PtrBBX1/2/5/8/23/24/25/29). Based on the relationship of homology and collinearity (Figure 9), PyunBBXs with high homology to these eight genes were selected from the BBX gene family of P. yunnanensis [12]. A total of 12 PyunBBX genes were obtained. In order to determine whether these 12 PyunBBX genes interact with MYB, PyunBBX genes with MYB binding sites were screened according to PlantCARE results, and finally, six PyunBX genes were determined ( Figure S3, Table S2). Under UV treatment, the results of RT-qPCR showed that the six PyunBBX genes showed different expression patterns (Figure 10), and the relative expression level of PyunBBX3/4/42 increased compared with the control group. The expression of PyunBBX12 was inhibited, suggesting that PyunBBX12 may be involved in the negative feedback regulatory pathway.
The expression level of PyunBBX18 was up-regulated on day 7, which was consistent with anthocyanins accumulation observed on day 7 (Figure 11), indicating that PyunBBX18 may be directly involved in regulating the anthocyanins biosynthesis pathway. Compared with the control group, the expression level of PyunBBX13 showed a high-low-high trend under UV treatment, indicating the complexity of PyunBBX13 response to UV.

Expression Patterns of Six PyunBBXs under UV Treatment by RT-qPCR Analysis
In order to determine the PyunBBX genes in response to UV, we picked out the highly expressed PtrBBXs of P. trichocarpa under the UV-B treatment (PtrBBX1/2/5/8/23/24/25/29). Based on the relationship of homology and collinearity (Figure 9), PyunBBXs with high homology to these eight genes were selected from the BBX gene family of P. yunnanensis [12]. A total of 12 PyunBBX genes were obtained. In order to determine whether these 12 PyunBBX genes interact with MYB, PyunBBX genes with MYB binding sites were screened 11), indicating that PyunBBX18 may be directly involved in regulating the anthocyanins biosynthesis pathway. Compared with the control group, the expression level of PyunBBX13 showed a high-low-high trend under UV treatment, indicating the complexity of PyunBBX13 response to UV. Figure 9. A rooted phylogenetic tree representing the relationships in P. yunnanensis, A. thaliana, and P. trichocarpa. We used the neighbor-joining (NJ) method with 1000 bootstrap replications to construct a phylogenetic tree. The BBX genes of a species are shown by genes name in the same colors and the same shapes in same colors (genes name in green and stars in red: PyunBBX genes of P. yunnanensis; genes name in orange and circles in yellow: PtrBBX genes of P. trichocarpa; genes name in purple and triangles in blue: AtBBX genes of A. thaliana). Figure 9. A rooted phylogenetic tree representing the relationships in P. yunnanensis, A. thaliana, and P. trichocarpa. We used the neighbor-joining (NJ) method with 1000 bootstrap replications to construct a phylogenetic tree. The BBX genes of a species are shown by genes name in the same colors and the same shapes in same colors (genes name in green and stars in red: PyunBBX genes of P. yunnanensis; genes name in orange and circles in yellow: PtrBBX genes of P. trichocarpa; genes name in purple and triangles in blue: AtBBX genes of A. thaliana).

Identification and Analysis of PyunBBX Gene Family Members in P. yunnanensis
In this study, 43 BBX gene family members were identified from the genome of P. yunnanensis, which can be divided into five types (Figure 3). They all have typical B-box structures, but the B-box1 structure of PyunBBX5 lacks the amino acid sequence of C-X2-C ( Figure S1). Therefore, PyunBBX5 is a type of BBX-like gene, and its functions need to be further verified.
The process of segmental and tandem duplication is crucial for the amplification of gene family members in the genome during evolution [53]. In this study, we analyzed tandem and segmental duplication events to study the evolutionary path of BBX genes in P. yunnanensis. We found only segmental duplication in BBX genes ( Table 2), suggesting that fragment duplication is involved in the expansion of PyunBBX genes.
In the phylogenetic tree ( Figure 7) of P. yunnanensis, P. trichocarpa, and A. thaliana, it was found that BBX family genes were not clustered strictly according to the combination of conserved domains, and the uneven distribution may be due to a large number of genes or slight differences in domain organization in plant species.
The promoter regions of 43 PyunBBX genes also contain cis-acting elements of various plant hormones, such as ABA (abscisic acid), JA (jasmonic acid), IAA (indoleacetic

Identification and Analysis of PyunBBX Gene Family Members in P. yunnanensis
In this study, 43 BBX gene family members were identified from the genome of P. yunnanensis, which can be divided into five types ( Figure 3). They all have typical B-box structures, but the B-box1 structure of PyunBBX5 lacks the amino acid sequence of C-X 2 -C ( Figure S1). Therefore, PyunBBX5 is a type of BBX-like gene, and its functions need to be further verified.
The process of segmental and tandem duplication is crucial for the amplification of gene family members in the genome during evolution [53]. In this study, we analyzed tandem and segmental duplication events to study the evolutionary path of BBX genes in P. yunnanensis. We found only segmental duplication in BBX genes ( Table 2), suggesting that fragment duplication is involved in the expansion of PyunBBX genes.
In the phylogenetic tree ( Figure 7) of P. yunnanensis, P. trichocarpa, and A. thaliana, it was found that BBX family genes were not clustered strictly according to the combination of conserved domains, and the uneven distribution may be due to a large number of genes or slight differences in domain organization in plant species.
In order to identify the UV-responsive PyunBBX genes under UV stress, we selected the up-regulated BBX genes of P. trichocarpa under UV treatment [12]. According to homology, we obtained 12 candidate PyunBBX genes. Finally, six PyunBBX genes were identified by the cis-acting elements of the promoter. The results of RT-qPCR showed that only the expression of PyunBBX3/4/18/42 was increased under UV treatment, while the expression of PyunBBX12 was inhibited. Moreover, PyunBBX13 showed functional complexity. The above shows that homologous genes of near-origin species may have similarities and differences in performing functions.
There is a VP motif binding to COP1 on the C segment of PyunBBX18, indicating that it interacts with COP1. In addition, PyunBBX12/42 also has a VP motif, revealing that they are important factors involved in light signal transduction and regulatory pathways.

BBX Is Involved in Anthocyanins Biosynthesis
Structural genes encoding key enzymes in anthocyanins biosynthesis are regulated by transcription factors. Widely studied TFs include the MYB gene family, bHLH gene family, WD40 gene protein, etc., which regulate anthocyanins biosynthesis by combining with functional elements in the promoter region of structural genes. For example, overexpression of SIMYB75 in tomatoes can increase anthocyanin content in fruits [54]. MYB TFs Aft can negatively regulate anthocyanins biosynthesis by binding to the promoter of negative regulator SlMYBATV [55]. Two bHLH TFs, JAF13 and AN1, in petunias can directly or indirectly bind with MYB transcription factor AN2 to activate DFR expression and promote anthocyanins biosynthesis [56,57]. The expression level of the StWD40 gene in potatoes was significantly up-regulated in red and purple tubers, suggesting that the up-regulated expression level of WD40 could promote anthocyanins accumulation [58].
Studies have shown that UV-B light can induce anthocyanin accumulation in plants. In apples, both MdHY5 and MdBBX22 could independently increase anthocyanin accumulation in the callus of apples under UV-B induction, and further investigation revealed that MdBBX22 and MdHY5 had interaction to jointly promote anthocyanin accumulation. In addition, both MdBBX24 and MdBBX33 interact with MdHY5 to regulate anthocyanins biosynthesis [13], suggesting that BBX-HY5 interaction is a universal regulatory module [8,59]. Recent studies have revealed that the interaction between HY5 and the BBX TF family affects anthocyanin accumulation, and B-box proteins respond to light induction and assist HY5 proteins in participating in transcriptional regulation of anthocyanin biosynthesis in plants [60]. Therefore, whether PyunBBX18 interacts with HY5 to affect anthocyanin accumulation of P. yunnanensis under UV-B stress requires further experimental verification.
Flavonoids are usually accumulated in leaves, roots, and bark [61,62]. By analyzing RNA-seq data of leaves and bark of P. yunnanensis, PyunBBX18 was found to be highly expressed in leaves and young bark, suggesting that PyunBBX18 may be involved in the synthesis of flavonoids. According to PlantCARE prediction results, the promoter of PyunBBX18 has three MYB binding sites ( Figure S3), so it may interact with MYB TFs to regulate the synthesis of flavonoids.

Conclusions
In this study, 43 PyunBBX genes were identified and analyzed at the genome-wide level. They were distributed unevenly on 15 chromosomes, and their evolutionary relationships, physical and chemical properties, gene structure, and conserved motifs were analyzed. Collinearity analysis revealed that there were 14 pairs of homologous genes in the P. yunnanensis BBX gene family, and these genes were purified and selected. In addition, RNA-seq showed that PyunBBX18 was highly expressed in leaves and younger bark. The results showed that PyunBBX18 may be a key gene in UV-induced anthocyanin biosynthesis through the determination of anthocyanins content and the expression of genes.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/genes13101811/s1, Figure S1: Amino acid sequences of conserved domains (B-box1/B-box2, CCT, and VP) of BBX genes family in P. yunnanensis; Figure S2: Sampling positions on the cuttings of P. yunnanensis. Figure S3: Cis-acting elements of six PyunBBX genes; Table  S1: Primers for RT-qPCR;  Data Availability Statement: Genomic data of P. yunnanensis can be obtained by contacting the corresponding author. The data that support the findings of this study are available from the corresponding author upon reasonable request.