*3.2. Phylogenetic Characterization of BAG Family Genes in Tomato*

A comprehensive search was conducted for plant lineages, including 30 representative monocotyledons, eudicotyledons, basal angiosperms, ferns, bryophytes, and algae. As a result, 485 putative genes were identified from 30 plants with BAG as the keyword in the phytozome13 database, and 292 putative genes encoding BAG proteins were further identified by batch CD search, which were divided into five different groups (Figure 1). By now, some functions of the BAG family members have been studied in Arabidopsis and rice [7,25]. To predict the function of SlBAG proteins, the evolutionary relationship among *Arabidopsis thaliana* (7 genes), *Oryza sativa* (8 genes), and *Solanum lycopersicum* (10 genes) was constructed using the MUSCLE and PhyML method. The phylogenetic tree showed that these BAG proteins were divided into two groups (I, II) (Figure 2). Group I contained 14 members (with four, five, and five members of Arabidopsis, rice, and tomato, respectively). Cluster II included eleven members (with three, three, and five members of Arabidopsis, rice, and tomato, respectively). This classification is basically similar to the previous analysis of BAG proteins in Arabidopsis [3,21].

#### *3.3. Exon–Intron Arrangement and Conserved Motifs Analysis*

Gene structure diagrams of *SlBAG* genes, including the presence of exon-intron components, were constructed by the Gene Structure Display Server (GSDS) (Figure 3A). As a result, new gene structural sequences were presented in this study in addition to *SlBAG7*, and *SlBAG9*, which were reported [24]. *SlBAG7* and *SlBAG9* were predicted to exhibit no introns and two *SlBAG* genes (*SlBAG1*, *SlBAG4*) contained one intron, whereas five *SlBAG* genes in group II exhibited three introns. Besides, several conserved motifs among the SlBAG proteins were calculated using the MEME, a tool used to discover motifs in a group of related DNA or protein sequences (Figure 3A). The BAG domain was analyzed using the HMMER web server (Figure 3B). Besides the BAG domain, all members of group II (SlBAG2, 5, 6, 8, 10) contained ubiquitin-like domains similar to some animal counterparts. However, SlBAG4, SlBAG7, and SlBAG9 of group I contained an IQ calmodulin-binding motif located in the N terminus, which was unique to plants. The previous study showed that the SlBAG4 has no IQ calmodulin-binding motif [24]. However, here we proved that its gene and protein structure were completely different from that in the existing database.

### *3.4. Organ-Specific Expression Profiles of SlBAG Genes*

The promoters of all ten *SlBAG* genes contained different cis elements for plant growth regulation, stress responses, hormone responses, and light responses (Figure S1; Table S3). To evaluate the function of *SlBAG* genes in the growth and development, the expression pattern of ten *SlBAG* genes in different organ (root, stem, leaves, flowers, green fruits, and

red fruits) was determined with qRT-PCR. The expression of *SlBAG* genes, shown as a cluster heat map, was different in various organs and some genes were only expressed in specific organs (Figure 4). *SlBAG2*, *SlBAG3*, *SlBAG4*, and *SlBAG5* were highly expressed in roots. *SlBAG8* displayed the high expression in roots and leaves. For flowers and fruits, *SlBAG6* showed the highest transcript level. *SlBAG7* showed relatively higher expression in flowers and green fruits. However, *SlBAG1*, *SlBAG9*, and *SlBAG10* showed the highest level in red fruits. These results suggest that *SlBAG* genes might be involved in the growth and development of tomato.

**Figure 1.** The number of BAG family in 30 plant species. The number of BAG proteins in 30 plant species and each group were shown on the right. The whole genomic data come from Phytozome 13. The number of BAG conserved domain analyzed by batch CD search were shown on the right. The species evolution tree was constructed by TimeTree 5 Beta (http://www.timetree.org, accessed on 10 January 2022).

**Figure 2.** Phylogenetic tree of tomato SlBAG proteins. The 25 amino acid sequences of three plant species (Sl, *Solanum lycopersicum*; Os, *Oryza sativa*; At, *Arabidopsis thaliana*) were generated using the PhyML in MEGA 7.0. Based on the phylogenetic data, these proteins are divided into two distinct sub-groups, which are distinguished with different colors.

**Figure 3.** Gene structure and BAG domain of SlBAG family in tomato. (**A**) The gene structure was generated using a Gene Structure Display Server (GSDS version 2.0). The length of exons as well as introns and UTRs of each *SlBAG* gene are displayed proportionally. (**B**) BAG domains were generated through the HMMER web server (Biosequence analysis using profile hidden Markov Models|HMMER). The legends of BAG domain, Ubiquitin-like domain and IQ calmodulin-binding motif are listed on the right. The phylogenetic tree was constructed referring to Figure 2. SlBAG proteins are listed on the left.

**Figure 4.** Expression profiles of *SlBAG* genes in different organs of tomato. The heatmap shows the expression change of ten *SlBAG* genes. Total RNA was extracted from different organs (root, stem, leaf, flower, green fruit, and red fruit) for qRT-PCR. Three independent biological repeats were performed (*n* = 3). The bar in the upper right corner represents the expression value of log2, and the change of expression level is represented by the change of color. Red indicates relatively high expression and blue indicates relatively low expression.
