Genome-Wide Characterization of B-Box Gene Family in Salvia miltiorrhiza

B-box (BBX) is a type of zinc finger transcription factor that contains a B-box domain. BBX transcription factors play important roles in plant photomorphogenesis, signal transduction, as well as abiotic and biological stress responses. However, the BBX gene family of Salvia miltiorrhiza has not been systematically investigated to date. For this study, based on the genomic data of Salvia miltiorrhiza, 27 SmBBXs genes were identified and clustered into five evolutionary branches according to phylogenetic analysis. The promoter analysis suggested that SmBBXs may be involved in the regulation of the light responses, hormones, stress signals, and tissue-specific development. Based on the transcriptome data, the expression patterns of SmBBXs under different abiotic stresses and plant hormones were analyzed. The results revealed that the expressions of the SmBBXs genes varied under different conditions and may play essential roles in growth and development. The transient expression analysis implied that SmBBX1, SmBBX4, SmBBX9, SmBBX20, and SmBBX27 were in the nucleus. A transcriptional activation assay showed SmBBX1, SmBBX4, SmBBX20, and SmBBX24 had transactivation activities, while SmBBX27 had none. These results provided a basis for further research on the role of SmBBXs in the development of Salvia miltiorrhiza.

BBX proteins are extensively involved in various plant growth and development processes [8], such as secondary metabolite synthesis, photomorphogenesis, flowering, signal transduction, abiotic and biological stress responses [9], etc. The MdBBX22-miR858-MdMYB9/11/12 module cooperates to regulate the accumulation of proanthocyanidins in apple peel [10]. In Malus pumila, MdBBX37 inhibits anthocyanin biosynthesis and promotes a hypocotyl elongation by negatively regulating the photosignaling pathway [11]. MdBBX37 integrates light, JA (jasmonic acid), ABA (abscisic acid), and ethylene signaling to regulate the leaf senescence by interacting with MdbHLH93, MdABI5, and MdEIL1 [12]. In Solanum lycopersicum, SlBBX20 activates the expression of SlDFR by binding to G-box1 in the promoter region of SlDFR, thus promoting anthocyanin biosynthesis [13]. The S. lycopersicum transcription factor module SlBBX20/21-SlHY5 regulates the photomorphogenesis of tomato [14]. The activities of AtCOP1 were inhibited under light exposure, resulting in the accumulation of AtBBX28 and AtHY5, where AtHY5 regulates the expression of many downstream target genes to promote photomorphogenesis [15]. BBX28 and BBX29 play important roles in facilitating the integration of light and brassinosteroid signals to regulate plant morphogenesis [16]. In A. thaliana, BBX16 induces and promotes photomorphogenesis under moderate light and is inhibited by GUN1/GLK1 following chloroplast damage [17]. The overexpression of AtBBX18 and AtBBX19 in A. thaliana can significantly prolong circadian cycles [18], whereas AtBBX7/AtBBX8 can positively regulate the frost resistance of plants [19]. Furthermore, the heterologous expression of CmBBX22 can delay leaf senescence in A. thaliana [20]. With deeper research, the molecular functions and regulatory networks of BBX in different plants have been revealed, which has attracted increasing attention to the field of plant molecular genetics.
S. miltiorrhiza is known as a model medicinal plant due to its small genome and perfect transgenic system, which has an exceptionally high therapeutic value [21][22][23]. However, the BBX gene family in S. miltiorrhiza has not yet been identified via functional analysis. We identified 27 SmBBXs genes based on the whole-genome data analysis of S. miltiorrhiza. From the cis-element analysis results, the homologous protein analysis of S. miltiorrhiza and A. thaliana, and the protein interaction prediction results, we selected SmBBX1/4/20/24/27 as the candidate genes for transcription activation experiments. Subsequently, according to previous reports [24][25][26][27][28], transcriptome data were selected to analyze the transcription levels of SmBBXs under various plant hormones (GA 3 and ABA) and stress treatments (NaCl and PEG). These results laid a foundation for a further study on the functional characteristics of BBX genes in S. miltiorrhiza.

Identification of SmBBXs Genes
Based on gene annotations and the conserved B-box motif characteristics of the BBX members, a total of 27 SmBBX genes were identified, with the detailed data for each SmBBX presented in Table 1. The amino acid (AA) lengths of the 27 SmBBXs ranged from 149 aa (SmBBX12) to 430 aa (SmBBX25), while the molecular weights of all proteins were 16-48 kD (SmBBX12 and SmBBX14). The Pl (isoelectric point) of all SmBBXs was lower than seven, which indicated that all SmBBXs were acidic proteins, with the most acidic being SmBBX13. Additionally, it was found that all SmBBXs were hydrophilic proteins, among which the SmBBX15 exhibited the greatest hydrophilicity. Except for SmBBX16, SmBBX19, and SmBBX22, most SmBBXs had instability indices of >40 and were therefore classified as unstable proteins. All SmBBXs were localized in the nucleus without signal peptides (Supplementary Figure S1) and transmembrane domains (Supplementary Figure S2). Table 1. Nomenclature, CDS, peptide lengths, molecular weights (MW), theoretical isoelectric points (PI), instability and aliphatic indices, Grand Average of Hydropathicity (GRAVY) and subcellular localization of Salvia miltiorrhiza BBX gene family.

Protein Sequences and Phylogenetic Analyses of SmBBXs
The domain logos and sequences of the B-box1, B-box2, and CCT domains of the SmBBX proteins are shown in Figure 1. Six members of the 27 SmBBXs were characterized by the occurrence of two B-box domains and a conserved CCT domain. Only two B-box domains were found in six SmBBXs, whereas eight members had one B-box domain and a CCT domain, and seven had only one B-box domain ( Table 2). Among the three domains, we found that the BBX motif contained ∼43 residues with the consensus sequence C-X2-C-X8-C-X7-C-X2-C-D-X3-H-X8-H-X4 ( Figure 1). Additionally, the consensus sequence of the conserved CCT domain was K-X2-R-Y-X2-R-K-X2-R-K-X2-A-X2-R-X-R-X-K-G-R-F ( Figure 1).
Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2). All SmBBX protein sequences were clustered into five subfamilies. In group 1 (except for SmBBX3, which had only one BBX and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 2 (except for SmBBX5, SmBBX6, SmBBX9, and SmBBX25, which had only one BBX domain and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 3, all SmBBXs had BBX and CCT domains. In group 4 (except for SmBBX19, which had only one BBX domain), all other SmBBXs had two BBX domains. In the group 5 (except for SmBBX6, which had both BBX and CCT domains), the other SmBBXs had only one BBX domain.

Analysis of cis-Elements in SmBBXs Promoter Region
A total of 63 major cis-elements were predicted in the SmBBXs gene promoter region ( Figure 3A), including 26 light-responsive, 13 hormone-responsive, 16 stress-responsive, and 8 tissue-specific and development-related elements. The number of light-responsive cis-elements was largest in the 26 SmBBXs gene promoters ( Figure 3A), wherein the total number of light-responsive elements was the largest, including G-box (27.5%), Box4 (26.6%), and GT1-motif (8.0%). The main stress responding cis-elements included STRE (25.5%), MYB (19.7%), and ARE (anaerobic cis-regulatory element) (15.6%). The main hormone responsive cis-elements included ABRE (involved in abscisic acid response) (23.0%), the AAGAA-motif (12.7%), the CGTCA-motif (methyl jasmonate-responsive cis-regulatory element) (12.4%), and the TGACG-motif (12.4%). The primary tissue-specific and development-related cis-elements included O2-site (involved in the regulation of the zein metabolism) (30.9%), CAT-box (meristem-specific expression elements) (20.0%), and CCGTCC-box (meristem-specific expression elements) (16.4%) ( Figure 3B). Our findings suggested that the promoter region that contained the SmBBXs gene played a critical role in the photonic and hormone responses.       Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2   Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2  Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2). All SmBBX protein sequences were clustered into five subfamilies. In group 1 (except for SmBBX3, which had only one BBX and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 2 (except for SmBBX5, SmBBX6, SmBBX9, and SmBBX25, which had only one BBX domain and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 3, all SmBBXs had BBX and CCT domains. In group 4 (except for SmBBX19, which had only one BBX domain), all other SmBBXs had two BBX domains. In the group 5 (except for SmBBX6, which had both BBX and CCT domains), the other SmBBXs had only one BBX domain.  Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2). All SmBBX protein sequences were clustered into five subfamilies. In group 1 (except for SmBBX3, which had only one BBX and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 2 (except for SmBBX5, SmBBX6, SmBBX9, and SmBBX25, which had only one BBX domain and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 3, all SmBBXs had BBX and CCT domains. In group 4 (except for SmBBX19, which had only one BBX domain), all other SmBBXs had two BBX domains. In the group 5 (except for SmBBX6, which had both BBX and CCT domains), the other SmBBXs had only one BBX domain.  Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2). All SmBBX protein sequences were clustered into five subfamilies. In group 1 (except for SmBBX3, which had only one BBX and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 2 (except for SmBBX5, SmBBX6, SmBBX9, and SmBBX25, which had only one BBX domain and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 3, all SmBBXs had BBX and CCT domains. In group 4 (except for SmBBX19, which had only one BBX domain), all other SmBBXs had two BBX domains. In the group 5 (except for SmBBX6, which had both BBX and CCT domains), the other SmBBXs had only one BBX domain.  Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2). All SmBBX protein sequences were clustered into five subfamilies. In group 1 (except for SmBBX3, which had only one BBX and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 2 (except for SmBBX5, SmBBX6, SmBBX9, and SmBBX25, which had only one BBX domain and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 3, all SmBBXs had BBX and CCT domains. In group 4 (except for SmBBX19, which had only one BBX domain), all other SmBBXs had two BBX domains. In the group 5 (except for SmBBX6, which had both BBX and CCT domains), the other SmBBXs had only one BBX domain.  Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2). All SmBBX protein sequences were clustered into five subfamilies. In group 1 (except for SmBBX3, which had only one BBX and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 2 (except for SmBBX5, SmBBX6, SmBBX9, and SmBBX25, which had only one BBX domain and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 3, all SmBBXs had BBX and CCT domains. In group 4 (except for SmBBX19, which had only one BBX domain), all other SmBBXs had two BBX domains. In the group 5 (except for SmBBX6, which had both BBX and CCT domains), the other SmBBXs had only one BBX domain.      Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2  Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2  Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2). All SmBBX protein sequences were clustered into five subfamilies. In group 1 (except for SmBBX3, which had only one BBX and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 2 (except for SmBBX5, SmBBX6, SmBBX9, and SmBBX25, which had only one BBX domain and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 3, all SmBBXs had BBX and CCT domains. In group 4 (except for SmBBX19, which had only one BBX domain), all other SmBBXs had two BBX domains. In the group 5 (except for SmBBX6, which had both BBX and CCT domains), the other SmBBXs had only one BBX domain.  Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2). All SmBBX protein sequences were clustered into five subfamilies. In group 1 (except for SmBBX3, which had only one BBX and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 2 (except for SmBBX5, SmBBX6, SmBBX9, and SmBBX25, which had only one BBX domain and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 3, all SmBBXs had BBX and CCT domains. In group 4 (except for SmBBX19, which had only one BBX domain), all other SmBBXs had two BBX domains. In the group 5 (except for SmBBX6, which had both BBX and CCT domains), the other SmBBXs had only one BBX domain.  Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2). All SmBBX protein sequences were clustered into five subfamilies. In group 1 (except for SmBBX3, which had only one BBX and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 2 (except for SmBBX5, SmBBX6, SmBBX9, and SmBBX25, which had only one BBX domain and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 3, all SmBBXs had BBX and CCT domains. In group 4 (except for SmBBX19, which had only one BBX domain), all other SmBBXs had two BBX domains. In the group 5 (except for SmBBX6, which had both BBX and CCT domains), the other SmBBXs had only one BBX domain.  Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2). All SmBBX protein sequences were clustered into five subfamilies. In group 1 (except for SmBBX3, which had only one BBX and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 2 (except for SmBBX5, SmBBX6, SmBBX9, and SmBBX25, which had only one BBX domain and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 3, all SmBBXs had BBX and CCT domains. In group 4 (except for SmBBX19, which had only one BBX domain), all other SmBBXs had two BBX domains. In the group 5 (except for SmBBX6, which had both BBX and CCT domains), the other SmBBXs had only one BBX domain.  Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2). All SmBBX protein sequences were clustered into five subfamilies. In group 1 (except for SmBBX3, which had only one BBX and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 2 (except for SmBBX5, SmBBX6, SmBBX9, and SmBBX25, which had only one BBX domain and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 3, all SmBBXs had BBX and CCT domains. In group 4 (except for SmBBX19, which had only one BBX domain), all other SmBBXs had two BBX domains. In the group 5 (except for SmBBX6, which had both BBX and CCT domains), the other SmBBXs had only one BBX domain.  Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2). All SmBBX protein sequences were clustered into five subfamilies. In group 1 (except for SmBBX3, which had only one BBX and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 2 (except for SmBBX5, SmBBX6, SmBBX9, and SmBBX25, which had only one BBX domain and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 3, all SmBBXs had BBX and CCT domains. In group 4 (except for SmBBX19, which had only one BBX domain), all other SmBBXs had two BBX domains. In the group 5 (except for SmBBX6, which had both BBX and CCT domains), the other SmBBXs had only one BBX domain.  Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2  Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2). All SmBBX protein sequences were clustered into five subfamilies. In group 1 (except for SmBBX3, which had only one BBX and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 2 (except for SmBBX5, SmBBX6, SmBBX9, and SmBBX25, which had only one BBX domain and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 3, all SmBBXs had BBX and CCT domains. In group 4 (except for SmBBX19, which had only one BBX domain), all other SmBBXs had two BBX domains. In the group 5 (except for SmBBX6, which had both BBX and CCT domains), the other SmBBXs had only one BBX domain.  Depending on the domain, we identified four distinct types of BBX proteins: SmBBXs with two B-boxes and one CCT domain, SmBBXs with one B-box and one CCT domain, SmBBXs with two B-boxes, and SmBBXs with one B-box domain. To better reveal the evolutionary relationships, we generated a phylogenetic tree with the known BBX families from Arabidopsis and Oryza sativa ( Figure 2). All SmBBX protein sequences were clustered into five subfamilies. In group 1 (except for SmBBX3, which had only one BBX and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 2 (except for SmBBX5, SmBBX6, SmBBX9, and SmBBX25, which had only one BBX domain and one CCT domain), all other SmBBXs had two BBX domains and one CCT domain. In group 3, all SmBBXs had BBX and CCT domains. In group 4 (except for SmBBX19, which had only one BBX domain), all other SmBBXs had two BBX domains. In the group 5 (except for SmBBX6, which had both BBX and CCT domains), the other SmBBXs had only one BBX domain.

Calculation of Ka/Ks Values for Homologous SmBBXs Gene Pairs
It was predicted that all Ka/Ks were less <1, which indicated that these SmBBXs proteins underwent a selective evolutionary purification and tended to be stable (Table 3). This was conducive to maintaining functional conservation in the gene families. Furthermore, there were significant variations in the Ka/Ks values between different groups, which implied that they were subject to various degrees of selection pressure and evolutionary rates. For example, the SmBBX7&SmBBX8 group had the highest Ka/Ks value, which translated to an expedited rate of evolution. In contrast, the SmBBX18&SmBBX19 group had the smallest Ka/Ks value, meaning that its amino acid sites were more conserved and less prone to change.

Gene Structure, Motifs, and Sequence Analysis of SmBBXs
To further understand the genetic structural characteristics of the SmBBXs gene, its introns and exons were analyzed, and the results are shown in Figure 4. The number of exons ranged from one to five, as did the number of introns. Based on the phylogenetic analysis, the SmBBXs gene family was divided into five subgroups. The gene members in the same subgroup had the same (or similar) numbers of exons (e.g., two exons and one intron in group 3 and four exons in group 2).

Figure 2.
Molecular phylogenetic analysis of SmBBXs protein in Salvia miltiorrhiza. The phylogenetic tree using MEGA X with the neighbor-joining (NJ) method. The number at the branch represents the confidence value obtained by 1000 bootstrap tests. Eighty-nine BBX proteins were divided into five subclasses represented by different colored clusters. Red, orange, blue, purple, and green clusters represent subclasses I, II, III, IV, and V, respectively. Red stars, violet circles, and teal triangles represent Salvia miltiorrhiza, Arabidopsis thaliana, and Oryza sativa, respectively.

Analysis of cis-Elements in SmBBXs Promoter Region
A total of 63 major cis-elements were predicted in the SmBBXs gene promoter region ( Figure 3A), including 26 light-responsive, 13 hormone-responsive, 16 stress-responsive, and 8 tissue-specific and development-related elements. The number of light-responsive cis-elements was largest in the 26 SmBBXs gene promoters ( Figure 3A), wherein the total number of light-responsive elements was the largest, including G-box (27.5%), Box4 (26.6%), and GT1-motif (8.0%). The main stress responding cis-elements included STRE (25.5%), MYB (19.7%), and ARE (anaerobic cis-regulatory element) (15.6%). The main hormone responsive cis-elements included ABRE (involved in abscisic acid response) (23.0%), the AAGAA-motif (12.7%), the CGTCA-motif (methyl jasmonate-responsive cis-regulatory element) (12.4%), and the TGACG-motif (12.4%). The primary tissue-specific and development-related cis-elements included O2-site (involved in the regulation of the zein metabolism) (30.9%), CAT-box (meristem-specific expression elements) (20.0%), and CCGTCC-box (meristem-specific expression elements) (16.4%) ( Figure 3B). Our findings suggested that the promoter region that contained the SmBBXs gene played a critical role in the photonic and hormone responses. The phylogenetic tree using MEGA X with the neighbor-joining (NJ) method. The number at the branch represents the confidence value obtained by 1000 bootstrap tests. Eighty-nine BBX proteins were divided into five subclasses represented by different colored clusters. Red, orange, blue, purple, and green clusters represent subclasses I, II, III, IV, and V, respectively. Red stars, violet circles, and teal triangles represent Salvia miltiorrhiza, Arabidopsis thaliana, and Oryza sativa, respectively.
Motifs play a role in protein-protein interactions and transcriptional regulation, where their diversity represents that of the protein composition. An online analysis of 27 SmBBXs genes using MEME revealed a total of 20 major motif sequences, among which motif4, motif1, and motif14 were the most widely distributed. Compared with the results of the analysis of the conserved motifs above, motif1 was intimately related to the formation of the B-box1 and B-box2 domains, whereas Motif2 was closely related to the CCT domains. SmBBX11, SmBBX22, SmBBX13, and SmBBX24 possess two motif-1 domains, while all others contain one motif-1. Motif2 exists in group 1, group 2, and group 3, and motif15 exists only in group 3.

Analysis of Protein-Protein Interactions in SmBBXs
OrthoVenn2 was employed to conduct a homology comparison between S. miltiorrhiza and A. thaliana, which identified 16 SmBBXs orthologous genes in A. thaliana (Table 4). According to the orthologous genes of A. thaliana, a SmBBX protein interaction network was developed using the STRING website ( Figure 5), with a focus on the interactions between HY5, COP1, and BBXs. In protein mutual mapping, AtBZS1/AtLZF1/AtBBX21/AtSTO, and COP1 form an interesting HY5 triangle.

Calculation of Ka/Ks Values for Homologous SmBBXs Gene Pairs
It was predicted that all Ka/Ks were less <1, which indicated that these SmBBXs proteins underwent a selective evolutionary purification and tended to be stable (Table 3). This was conducive to maintaining functional conservation in the gene families. Furthermore, there were significant variations in the Ka/Ks values between different groups, which implied that they were subject to various degrees of selection pressure and evolutionary rates. For example, the SmBBX7&SmBBX8 group had the highest Ka/Ks value, which translated to an expedited rate of evolution. In contrast, the SmBBX18&SmBBX19 group had the smallest Ka/Ks value, meaning that its amino acid sites were more conserved and less prone to change.

Analysis of Protein-Protein Interactions in SmBBXs
OrthoVenn2 was employed to conduct a homology comparison between S. miltiorrhiza and A. thaliana, which identified 16 SmBBXs orthologous genes in A. thaliana (Table  4). According to the orthologous genes of A. thaliana, a SmBBX protein interaction network

Analysis of GO and KEGG in SmBBXs
In order to further explore the function of SmBBXs, GO analysis and KEGG analysis are performed (Figure 6). At the biological process level, SmBBXs were primarily enriched for cellular (GO:0009987), metabolic (GO:0008152), and single-organism processes (GO:0044699). At the molecular function level, SmBBXs were enriched for their catalytic activities (GO:0003824), binding (GO:0005488), and molecular function regulation (GO:0098772). At the cellular component level, SmBBXs were primarily enriched for their cells (GO:0005623), cell components (GO:0044464), and organelles (GO:0043226). KEGG predicted that the major enriched metabolic pathways in SmBBXs were primarily involved in the lipid metabolism, as well as global and overview maps.

Analysis of GO and KEGG in SmBBXs
In order to further explore the function of SmBBXs, GO analysis and KEGG analysis are performed (Figure 6). At the biological process level, SmBBXs were primarily

Subcellular Localization and Transcription Activation of SmBBX Genes
According to the amino acid sequences of the SmBBXs family genes, the Plant-mPLoc online tool was utilized to predict the subcellular localization. We forecasted that 27 SmB-BXs were localized in the nucleus, which indicated that the subcellular localization and differentiation of the SmBBXs family proteins were highly conserved. To obtain the subcellular localization of SmBBXs, those of SmBBX1, SmBBX4, SmBBX9, SmBBX20, and SmBBX27 were investigated by the transient expression in onion epidermal cells using a SmBBX transgene fused to a green fluorescent protein (GFP) driven by a CaMV35S promoter. The empty vector was used as the control, with the results showing that it was distributed throughout the cell. This experiment revealed that the SmBBX-GFP activity was located in the nucleus (Figure 7), which was consistent with the bioinformatics prediction (Table 1). Figure 7. Subcellular localization analysis. All candidate genes were independently cloned into vector pEarleyGate103. Subcellular localization of SmBBX1-GFP, SmBBX4-GFP, SmBBX9-GFP, SmBBX20-GFP, and SmBBX27-GFP in the nucleus was confirmed in onions via laser confocal microscopy. Scale bars = 100 μm.

Subcellular Localization and Transcription Activation of SmBBX Genes
According to the amino acid sequences of the SmBBXs family genes, the Plant-mPLoc online tool was utilized to predict the subcellular localization. We forecasted that 27 SmBBXs were localized in the nucleus, which indicated that the subcellular localization and differentiation of the SmBBXs family proteins were highly conserved. To obtain the subcellular localization of SmBBXs, those of SmBBX1, SmBBX4, SmBBX9, SmBBX20, and SmBBX27 were investigated by the transient expression in onion epidermal cells using a SmBBX transgene fused to a green fluorescent protein (GFP) driven by a CaMV35S promoter. The empty vector was used as the control, with the results showing that it was distributed throughout the cell. This experiment revealed that the SmBBX-GFP activity was located in the nucleus (Figure 7), which was consistent with the bioinformatics prediction (Table 1).

Subcellular Localization and Transcription Activation of SmBBX Genes
According to the amino acid sequences of the SmBBXs family genes, the Plant-mPLoc online tool was utilized to predict the subcellular localization. We forecasted that 27 SmB-BXs were localized in the nucleus, which indicated that the subcellular localization and differentiation of the SmBBXs family proteins were highly conserved. To obtain the subcellular localization of SmBBXs, those of SmBBX1, SmBBX4, SmBBX9, SmBBX20, and SmBBX27 were investigated by the transient expression in onion epidermal cells using a SmBBX transgene fused to a green fluorescent protein (GFP) driven by a CaMV35S promoter. The empty vector was used as the control, with the results showing that it was distributed throughout the cell. This experiment revealed that the SmBBX-GFP activity was located in the nucleus (Figure 7), which was consistent with the bioinformatics prediction (Table 1).  The SmBBXs were further fused to the DNA binding domain (BD) to investigate the transcription activation in the yeast cells. Both the negative control pGBKT7 construct and pGBKT7-SmBBX27 were unable to grow on the SD/-Trp-His-Ade that contained X-αgal medium, whereas the pGBKT7-SmBBX1, pGBKT7-SmBBX4, pGBKT7-SmBBX20, and pGBKT7-SmBBX24 constructs grew quite well on both media. According to the results (Figure 8), the SmBBX1 and SmBBX4 exhibited a more robust transcription activation, while SmBBX27 did not.
The SmBBXs were further fused to the DNA binding domain (BD) to investigate the transcription activation in the yeast cells. Both the negative control pGBKT7 construct and pGBKT7-SmBBX27 were unable to grow on the SD/-Trp-His-Ade that contained X-α-gal medium, whereas the pGBKT7-SmBBX1, pGBKT7-SmBBX4, pGBKT7-SmBBX20, and pGBKT7-SmBBX24 constructs grew quite well on both media. According to the results (Figure 8), the SmBBX1 and SmBBX4 exhibited a more robust transcription activation, while SmBBX27 did not.

Expression Profiles of SmBBXs Genes
According to the FPKM (Fragments per Kilobase million) values of the SmBBXs family genes in the transcriptome sequencing results, the expressions of the SmBBXs family genes under root, stem, leaf, flower, and abiotic stress were analyzed. TBtools was used to plot the heat map.
The expressions of the SmBBXs genes were different for the various organs of Salvia miltiorrhiza ( Figure 9A). SmBBX27, SmBBX1, SmBBX13, SmBBX16, SmBBX18, SmBBX23, SmBBX19, SmBBX26, SmBBX24, SmBBX11, and SmBBX17 had high expression levels in the roots, stems, leaves, and flowers of Salvia miltiorrhiza. The expression levels of SmBBX4, SmBBX8, SmBBX6, SmBBX12, SmBBX15, SmBBX7, SmBBX3, SmBBX2, SmBBX25, and SmBBX9 in all tissues were low. SmBBX5, SmBBX14, SmBBX20, and SmBBX22 were negligibly expressed in all tissues. This indicated that the expression specificities of SmBBXs genes in different tissues were related to their various functions. To confirm whether the expressions of SmBBXs were affected by different stressors (based on the cis-element analysis results of SmBBXs and the literature reports), the transcriptome data of the four treatments (GA 3, ABA, PEG, and NaCl) were utilized to analyze the transcript abundance.

Discussion
As a class of zinc finger proteins, BBX transcription factors are extensively involved in various plant growth and development processes. They are engaged in many light signal pathways in plants and have certain functions against biotic and abiotic stress. The roles of BBX genes in medicinal plants have rarely been studied. However, they are involved in light morphogenesis, flower development, shade avoidance effects, plant signal transduction, and abiotic and biotic stress responses, which are also critical for the production and breeding of medicinal plants. Therefore, it is necessary to further investigate the functions of BBX genes in essential medicinal plants. In this study, the members of the BBX transcription factor family of Salvia miltiorrhiza were identified by BLAST alignment and conserved domain analysis. A total of 27 BBXs transcription factors were identified from the entire genome of S. miltiorrhiza, and their phylogenetic trees, cis-element, gene structures, subcellular localizations, autoactivation functions, hormone treatments, and stress tolerance expression patterns under abiotic stress were analyzed.
Numerous photoresponsive elements (e.g., Box4, G-box, and CTT-motifs) were distributed on the SmBBXs promoter sequence. Hormone response elements included the ABRE (abscisic acid response element), the TCA-element (salicylic acid response element), the TATC-box (gibberellin response element), the CGTCA-motif (jasmonic acid response element), etc. The abiotic stress response elements included the MBS (salt stress response elements and drought-induced MYB transcription factor binding sites), DRE (against high salt stress), and TC-rich repeats (against drought stress and pest stress). The Cis-element analysis indicated that the SmBBXs family might play an important role in light signals, hormones, and abiotic stresses. For example, SmBBX4 is highly expressed in leaves but minimally expressed in other tissues, while SmBBX17 is highly expressed in all tissues, indicating that the functions of SmBBXs vary in different tissues.
For this study, transcriptome data were used to further analyze the expressions of SmBBXs under GA 3 , ABA, salt stress, and drought treatments, with the results revealed that most SmBBXs were responsive. It was observed that CmBBX19 and ABF3 complexes can regulate plant drought tolerance through an ABA-dependent pathway [29]. The overexpression of AtBBX5 enhances the salt tolerance of transgenic Arabidopsis through an ABA-dependent pathway [30], while SmBBX11 (a direct homolog of AtBBX5) is differentially expressed under salt stress and an ABA treatment. SmBBX11 may function similarly to AtBBX5 and participate in the salt-stress responses through an ABA-dependent pathway.
The two BBX conserved domains of SmBBXs transcription factors are very similar to those of Oryza sativa BBX and pepper BBX. The CCT domain of SmBBXs is similar to the CCT domain of pepper, indicating that BBX and CCT domains are strongly conserved between species. In SmBBXs, the sequence identity of the B-box1 domain was 58.75%, while that of the B-box2 domain was 58.33%, and that of the CCT domain was 73.21%; thus, the three SmBBXs domains were conserved. Conserved domains play significant roles in mediating protein interactions and regulating the gene expression. The B-box domain is thought to be required for interactions with HY5 and transcriptional regulation, as the AtBBX25 functions by interacting with HY5 through its B-boxes. The second B-box in AtSTH3 is important for HY5-STH3 interactions. AtBBX24 can form a heterodimer with HY5, which interferes with the binding of HY5 to the anthocyanin biosynthesis gene promoter, and thus inhibits their expressions [31]. PpBBX18 forms heterodimers with PpHY5 through two B-box domains and induces PpMYB10 transcription [32]. PtrHY5 enhances the activation of PtrBBX23 to downstream genes by interacting with the second B-box at the N-terminal of the PtrBBX23 gene through the C-terminal bZIP domain [33].
Anthocyanins belong to biological flavonoids, which are a type of water-soluble natural pigment. For plants, anthocyanins serve as natural photoprotectants that can remove oxygen free radicals to protect them from strong light burns, absorb excess visible light, protect against UV rays, and help plants resist biotic and abiotic stresses [34]. In the human body, anthocyanins confer potent antioxidant capacities, which have an important medical value (e.g., anti-tumor, anti-aging, anti-fatigue, the regulation of intestinal flora, and the lowering of blood lipids) [35,36]. As a model medicinal plant, it is of great significance for studying the synthetic kinetics of anthocyanins. In A. thaliana, BBXs proteins were involved in the photomorphogenesis of seedlings and photoinduction responses and formed a common regulatory module with COP1 and HY5 proteins [37,38] to participate in the transcriptional regulation of anthocyanin synthesis in plants [39]. In the dark, COP1 mediates the degradation of BBX and HY5 via the 26S proteasome system to promote dark morphogenesis. Under light exposure, COP1s activity is inhibited in a photo dependent manner, which allows BBX and HY5 to accumulate to promote photomorphogenesis. BBX interacts with HY5, interferes with the binding of HY5 to the target, and inhibits the transcriptional activity of HY5, thus negatively regulating photomorphogenesis. Based on the mapping of the A. thaliana protein interaction network, it was speculated that SmBBX20 might also regulate anthocyanin synthesis through a co-regulation with COP1 and HY5 [40]. At the genome-wide level, we analyzed the direct homologs of A. thaliana and S. miltiorrhiza genes and selected 16 direct homologs of SmBBXs from A. thaliana to perform interactive protein mapping to plot the potential functions of SmBBXs. In protein mutual mapping, AtBZS1/AtLZF1/AtBBX21/AtSTO and COP1 form an interesting HY5 triangle. We hypothesized that SmBBX20/SmBBX21/SmBBX23/SmBBX24, COP1, and HY5 proteins comprise a common control module which is involved in the transcriptional regulation of anthocyanin synthesis in S. miltiorrhiza. This will need to be verified through subsequent experiments.
Since eukaryote transcription processes primarily occur in the nucleus, this is typically where transcription factors are found. According to the phylogenetic tree, 27 SmBBXs were divided into five groups, and one gene was selected from each of them to verify the subcellular localization results (Figure 7). The experimental results were consistent with the prediction, as the SmBBX1/SmBBX4/SmBBX9/SmBBX20/SmBBX27 localization in the nucleus revealed them as transcription factors with regulating roles in the cell nucleus.

Sequence Alignment and Phylogenetic Analysis
The phylogenetic tree can visually display the homologic relationships between genes. Thus, to determine the evolutionary relationships between BBXs genes, phylogenetic trees for AtBBXs, OsBBXs, and SmBBXs proteins were developed with MEGA X software [45] using the neighbor-joining method (NJ). Test of phylogeny: the bootstrap method, No. of bootstrap replications: 1000, model/method: p-distance, rates between sites: uniform rates, gaps/missing data treatment: pairwise deletion, and number of threads: 7. Evolutionary tree beautification was performed using the online website Evolview (http://www.evolgenius.info/evolview/, accessed on 6 April 2022). The sequences of the three conserved regions of SmBBXs BBX1, BBX2, and CCT were multiply aligned using ClustalW [46], and the sequence identification was constructed online (http://weblogo.berkeley.edu/logo.cgi, accessed on 6 April 2022).
For this study, the diversity of the SmBBXs protein motif compositions was investigated using the MEME online website (https://meme-suite.org/meme/tools/meme, accessed on 6 April 2022) [48]. The maximum number of motifs was set to 8, and the motif length was set to 6-200 amino acids, while other parameters were set by default. Tbtools v1.089 software (Chengjie Chen et al., China) [49] was used to visualize the intron and exon structures of the SmBBXs gene. This study also employed PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/, accessed on 6 April 2022) online software to predict the secondary structures of the SmBBXs protein.

Cis-Element Analysis for BBX Gene Promoters
To analyze the potential tissue-specific and developmental hormone responses, stress responses, and light response-related cis-elements of the SmBBXs gene, the 2000 bp up-stream (ATG) promoter sequences of the 27 SmBBXs gene initiation codons were employed using the online PlantCARE website [50], to predict the cis-elements of SmBBXs and count the light-responsive, hormone-responsive, stress-responsive, tissue-specific, and developmental-related elements.

Calculation of Ka/Ks Values for Homologous SmBBXs Gene Pairs
The Ka and Ks values of the homologous genes were calculated using PAL2NAL (http://www.bork.embl.de/pal2nal/, accessed on 6 April 2022) to predict how they were evolutionarily selected. Among them, when Ka/Ks > 1, the gene was considered to be under a positive selective pressure, more likely to produce non-synonymous mutations and cause changes in the amino acid sequence. When Ka/Ks = 1, the gene was considered to be under neutral selective pressure, whereas when Ka/Ks < 1, it was considered to be under negative selective pressure and more inclined to produce synonymous mutations, thereby maintaining the stability.

Gene Ontology (GO) and KEGG Annotation
GO analysis was performed on the SmBBXs family, which encompassed the biological processes, cellular components, and molecular functions. Enrichment was intuitively displayed through the distribution of differential genes on the GO enrichment histogram, which facilitated molecular analysis. To elucidate the various levels of protein functionality, KEGG was employed to analyze the metabolic and transduction pathways in which the SmBBXs gene family was primarily involved in the systematic study of the SmBBXs functional and expression data. GO and KEGG analysis were performed using the online website (https://www.omicshare.com/tools/, accessed on 6 April 2022).

Protein Interaction Network Analysis
Orthologous genes are derived from a common ancestor, and when those between species exhibit a high degree of sequence similarity, they tend to have similar biological functions [51]. As a model plant, A. thaliana protein interactions are accurate and comprehensive. Therefore, the orthovenn2 website (https://orthovenn2.bioinfotoolkits.net/home, accessed on 6 April 2022) [52] was used to search for the orthologous genes of A. thaliana and S. miltiorrhiza. Further, a sequence comparison was conducted to verify the sequence similarity so as to screen genes that may have similar functions to SmBBXs in the AtBBXs. We used the website String10 (http://string-db.org/, accessed on 6 April 2022) to analyze the protein interaction network of AtBBXs and map the protein network of SmBBXs. The minimum required interaction score was set as high confidence (0.7). Finally, Cytoscape 3.7 software was used to map the protein interaction network.

Subcellular Localization of BBX Proteins
Transcription factors often play key roles in cell nuclei; thus, BBX proteins were located in the nucleus as predicted. To confirm their localization, we developed a pEarleyGate103-SmBBX1/4/9/20/27 vector connected with full-length BBX open reading frames (ORFs) and gene sequences without termination codons using the gateway method. Through biolistic PDS-1000/He, we transformed the plasmid DNA into 3 × 3 cm young onion endocuticle cells, after which the transient expression of SmBBX-GFP was observed using a fluorescence microscope (Leica DM6000B, Wetzlar, Germany) and the nucleus was visualized using DAPI.

Transactivation Activity Assay of SmBBXs Genes
The ORFs (open reading frames) of SmBBX1, SmBBX4, SmBBX20, SmBBX24, and SmBBX27 were cloned into the pGBKT7 vector using the gateway method to generate the pGBKT7-SmBBX recombinant vector, which was then transformed with the empty vector into the yeast strain AH109, respectively. The transformants carrying the pGBKT7-SmBBXs and empty pGBKT7 (negative control) were cultured on a SD/-Trp solid medium, and once the yeast successfully transformed to the pGBKT7-SmBBXs plasmid, they were expanded with SD/-Trp liquid and diluted to 1, 1/10, 1/100, and 1/1000, which were inoculated (5 µL each) on a SD/-Trp-His-Ade solid medium that contained 5 mg/mL X-α-gal. Proteins with transcriptional activities grew normally on the SD/-Trp-His-Ade solid medium containing 5 mg/mL X-α-gal and turned blue. If the yeast colony did not grow, this meant that the target protein had no transcriptional activation capacity.

Expression Analysis of SmBBXs Genes Based on Transcriptome Sequencing
Transcriptome sequencing was performed on four different tissues (root, stem, leaf, and flower) of S. miltiorrhiza, and the expression patterns of SmBBXs were further detected. GA-, ABA-, NaCl-, and PEG-treated S. miltiorrhiza were collected at time points for transcriptome sequencing to further detect the change in the SmBBXs expression level. The method for calculating the SmBBXs transcription abundance is estimated in terms of the fragments per kilobase per million mapped fragments (FPKM) [53].
According to the FPKM values of the SmBBXs genes in the transcriptome sequencing results, the expressions of SmBBXs genes under the root, stem, leaf, flower, and abiotic stress were analyzed, and TBtools v1.089 software (Chengjie Chen et al., China) [49] was employed to draw a heat map.

Conclusions
In this study, 27 SmBBXs genes were identified and analyzed at the genome-wide level for S. miltiorrhiza, which found that SmBBXs and AtBBXs contained 16 orthologous genes, with which the protein interaction network was constructed to analyze the potential SmBBXs functions. Transcriptome data were employed to analyze the expression patterns of SmBBXs under abiotic stress and different S. miltiorrhiza hormone treatments. Additionally, a transactivation activity assay and the subcellular localization of SmBBXs were analyzed. The results of this study lay a foundation for further research on the functions of SmBBXs under different abiotic stresses, which is of a great significance for the production and breeding of medicinal plants.