Identification and Characterization of Tomato SWI3-Like Proteins: Overexpression of SlSWIC Increases the Leaf Size in Transgenic Arabidopsis

As the subunits of the SWI/SNF (mating-type switching (SWI) and sucrose nonfermenting (SNF)) chromatin-remodeling complexes (CRCs), Swi3-like proteins are crucial to chromatin remodeling in yeast and human. Growing evidence indicate that AtSWI3s are also essential for development and response to hormones in Arabidopsis. Nevertheless, the biological functions of Swi3-like proteins in tomato (Solanum lycopersicum) have not been investigated. Here we identified four Swi3-like proteins from tomato, namely SlSWI3A, SlSWI3B, SlSWI3C, and SlSWI3D. Subcellular localization analysis revealed that all SlSWI3s are localized in the nucleus. The expression patterns showed that all SlSWI3s are ubiquitously expressed in all tissues and organs, and SlSWI3A and SlSWI3B can be induced by cold treatment. In addition, we found that SlSWI3B can form homodimers with itself and heterodimers with SlSWI3A and SlSWI3C. SlSWI3B can also interact with SlRIN and SlCHR8, two proteins involved in tomato reproductive development. Overexpression of SlSWI3C increased the leaf size in transgenic Arabidopsis with increased expression of GROWTH REGULATING FACTORs, such as GRF3, GRF5, and GRF6. Taken together, our results indicate that SlSWI3s may play important roles in tomato growth and development.


Introduction
The fundamental unit of chromatin is the nucleosome, which is composed of two turns of DNA wrapped around a histone octamer (two H2A-H2B dimers and one H3-H4 tetramer) [1]. To readout genomic information, a dynamic chromatin environment is needed during the cellular processes such as transcription, DNA replication and recombination. The modification of chromatin can occur through multiple mechanisms, including nucleosome composition and positioning by ATP-dependent chromatin-remodeling complexes (CRCs) and enzyme complexes that modify DNA or chromatin proteins [2,3]. CRCs use energy from ATP hydrolysis [4] and change local chromatin structure, thus enriching accessibility of transcription factors and availability of genomic information [5].
The first CRC, SWI/SNF CRC, was identified in two independent screens for mutants affecting mating-type switching (SWI) and growth on sucrose (sucrose nonfermenting, SNF) in yeast [6,7]. Biochemical analysis indicated that yeast SWI/SNF CRC is composed of 12 subunits [8], and the core complex including SWI2/SNF2-type ATPase, one SNF5, and two copies of SWI3 subunits, is sufficient for execution of nucleosome remodeling in vitro [9]. Moreover, several accessory subunits associating with the core complex act as an interface for interactions with other auxiliary proteins that affect chromatin remodeling activity [10].
As part of SWI/SNF CRC, yeast Swi3p is essential for the assembly of this complex, ATP-dependent H2A-H2B displacement and recruitment to target genes [11,12]. Further genome analysis indicated that Swi3-like proteins are found in virtually all eukaryotes, such as Drosophila, mammals and Arabidopsis. Intriguingly, studies in both yeast and mammalian cells showed that Swi3p and its mammalian homologs BAF155 and BAF170 can target many genes and genomic locations in the absence of Swi2 and other components of the SWI/SNF CRC complex [13,14], suggesting that they may have unique functions in gene regulation. Generally, all Swi3-like proteins contain two typical domains, the SWIRM and SANT domains. The SWIRM domain (Swi3p, Rsc8p, and Moira) consists of 85 amino acid residues and forms a compact helix-turn-helix (HTH)-related structure [15], while the SANT (SWI3, ADA2, N-CoR and TFIIIB) domain is structurally related to the homeodomain and the Myb DNA-binding domain [16]. Furthermore, in addition to Swi3-like proteins, the SWIRM domain is also found in LSD1 (Lysine-specific demethylase 1), Ada2 (Adenosine deaminase isoenzymes 2), and in a JAB domain-containing protein involved in protein degradation through the ubiquitin pathway [15]. Functional analysis demonstrated that SANT domains tether to both DNA and proteins and are essential for histone acetyltransferase activity [16,17].
In Arabidopsis, four Swi3-like proteins, namely CHB1 (AtSWI3A), CHB2 (AtSWI3B), CHB3 (AtSWI3C) and CHB4 (AtSWI3D), have been identified [18,19]. Current data show that they operate as modifiers of transcriptional or epigenetic regulation in plant growth and development. For example, mutations of AtSWI3A and AtSWI3B result in disruption of embryo development at the globular stage, while mutations in AtSWI3C and AtSWI3D display severe dwarfism, abnormal vegetative development, and reduced fertility [20]. Moreover, yeast-two-hybrid assays demonstrated that AtSWI3A and AtSWI3B can form homodimers and heterodimers, and also interact with BSH/SNF5, AtSWI3C, and the flowering regulator FCA, while AtSWI3D can only bind AtSWI3B [18,20]. Interestingly, when expressed in Saccharomyces cerevisiae, AtSWI3B can partially complement the swi3 mutant phenotype [18]. In addition to interacting with other SWI3 subunits, AtSWI3B also interacts with HYPERSENSITIVE TO ABA1 (HAB1), a negative regulator of ABA signaling, indicating that SWI3B is involved in the ABA signaling [21]. Subsequent research found that AtSWI3B physically interacts with IDN2, a lncRNA-binding protein, and contributes to lncRNA-mediated transcriptional silencing [22]. Recent data demonstrated that AtSWI3B (as well as AtSWI3C and AtSWI3D) interacts with MORC6 (MICRORCHIDIA 6) and a SET domain-containing histone methyltransferase SUVH9 (SU(VAR)3-9 homolog) to mediate transcriptional silencing [23].
In leaf development, the expression of IAMT1 (IAA Carboxyl Methyltransferase 1) is regulated by AtSWI3B-mediated chromatin remodeling [24]. In addition to auxin and ABA, the Arabidopsis Swi3-like proteins are also involved in other hormone signaling. For instance, the AtSWI3C mutation down-regulates the expression of GID1 GA receptor genes, affecting the GA perception in leaves [25]. Meanwhile, AtSWI3C physically interacts with several DELLA proteins, indicating that chromatin remodeling mediated by AtSWI3C may be required for the DELLA-mediated effects, such as activation of GID1 [25]. Moreover, the AtSWI3C (as well as AtSWI3D)-containing SWI/SNF chromatin remodeling complex is recruited by the transcriptional coactivator AN3 (ANGUSTIFOLIA3) to the promoters of leaf development related genes [26]. Consistently, modification of the AtSWI3C expression increases leaf size by increasing cell number but not cell size [26]. Moreover, recent data indicated that ZmCHB101, one of three maize SWI3D genes, plays essential roles in maize growth and development at both vegetative and reproductive stages [27], since the ZmCHB101 RNA interference plant lines displayed abaxially curling leaf and impaired tassel and cob development phenotypes.
Compared to Arabidopsis, the functions of Swi3-like proteins are largely unknown in other plant species. Nevertheless, phylogenetic analysis indicated that the Swi3-like proteins are widely present in the genomes of other plant species such as rice (Oryza sativa), Medicago truncatula and Zea mays [28]. In this study, we identified and characterized four Swi3-like proteins, namely SlSWI3A, SlSWI3B, SlSWI3C, and SlSWI3D, in tomato (Solanum lycopersicum). The expression profiles and subcellular localization of tomato Swi3-like proteins were investigated. In addition, the interaction of Swi3-like proteins with RIN and SlCHR8, two proteins involved in reproductive development, was also explored. Furthermore, ectopic expression of SlSWI3C in Arabidopsis resulted in the increased leaf size. Taken together, our results shed light on the potential functions of Swi3-like proteins during tomato development.

Identification and Phylogenetic Analysis of Tomato Swi3-Like Proteins
The Swi3-like proteins sequences of Arabidopsis, rice, yeast and human were used as queries to search against the SGN annotation database with the BLAST program. All sequences with an E-value below 10 −2 were selected for further analysis. Pfam and SMART databases were used to confirm each candidate protein sequence. Like Arabidopsis, four Swi3-like proteins (namely SlSWI3A, SlSWI3B, SlSWI3C, and SlSWI3D) were identified in the tomato genome ( Table 1). The open reading frames (ORFs) of SlSWI3s ranged from 1470 to 2838 bp, and the length of SlSWI3 proteins varied from 490 to 946 amino acids. Further bioinformatics analysis indicated that SlSWI3 proteins were potentially localized in different organelles including the nucleus, cytoplasm, chloroplast and mitochondria (Table 1).
To further investigate the evolutionary relationships of SlSWI3, we carried out phylogenetic analyses using Swi3-like proteins of different species. The phylogenetic tree indicated that the plant Swi3-like proteins can be clearly divided into four groups: SWI3A, SWI3B, SWI3C, and SWI3D, while the SWI3A/SWI3B pairs are more related to the branches of yeast and animal Swi3 sequences ( Figure 1A). The conserved domain analyses showed that all Swi3-like proteins contain two characteristic domains: SWIRM and SANT domains ( Figure 1B). Moreover, the SWIRM-assoc_1 (SWIRM-associated region 1) domain (Interpro: IPR032451), which was previously identified as the leucine zipper domain [20], was found in all members of Swi3-like proteins except for SlSWI3B ( Figure 1B). Like the AtSWI3D and OsSWI3Ds, a ZnF_ZZ domain (Interpro: IPR000433), which was named because of the ability to bind two zinc ions [29], was also found in the N-terminus of SlSWI3D. In general, ZZ-type zinc finger domains contained 4-6 Cys residues that participate in zinc binding as well as protein-protein interaction [30]. LOC_Os04g01970 XP_015633469 2661 887 nucl, chlo, mito 7 Yes associated region 1) domain (Interpro: IPR032451), which was previously identified as the leucine zipper domain [20], was found in all members of Swi3-like proteins except for SlSWI3B ( Figure 1B). Like the AtSWI3D and OsSWI3Ds, a ZnF_ZZ domain (Interpro: IPR000433), which was named because of the ability to bind two zinc ions [29], was also found in the N-terminus of SlSWI3D. In general, ZZ-type zinc finger domains contained 4-6 Cys residues that participate in zinc binding as well as protein-protein interaction [30].

Subcellular Localization of Tomato Swi3-Like Proteins
Bioinformatics analysis showed that SlSWI3 proteins exhibit various patterns of subcellular localization (Table 1). Nevertheless, a previous study demonstrated that Arabidopsis AtSWI3B localizes at the nucleus [21]. To determine the subcellular localization of tomato Swi3-like proteins, we performed in vivo targeting experiments in tobacco (N. benthamiana). To this end, SlSWI3A, SlSWI3B SlSWI3C and SlSWI3D were subcloned into the pEAQ-GFP vector to generate 35S: SlSWI3A-GFP, 35S: SlSWI3B-GFP, 35S: SlSWI3C-GFP and 35S: SlSWI3D-GFP constructs, respectively. The constructs were introduced into leaf cells of tobacco by A. tumefaciens infiltration [31]. The fluorescence was visualized through a laser-scanning confocal microscope, and DAPI staining was used to visualize nuclei localization. We found that the N. benthamiana epidermal cells exhibited strong fluorescence in the nuclei ( Figure 2, Figure S1), suggesting that the tomato SlSWI3-like proteins are nuclear proteins. GFP, 35S: SlSWI3B-GFP, 35S: SlSWI3C-GFP and 35S: SlSWI3D-GFP constructs, respectively. The constructs were introduced into leaf cells of tobacco by A. tumefaciens infiltration [31]. The fluorescence was visualized through a laser-scanning confocal microscope, and DAPI staining was used to visualize nuclei localization. We found that the N. benthamiana epidermal cells exhibited strong fluorescence in the nuclei ( Figure 2, Figure S1), suggesting that the tomato SlSWI3-like proteins are nuclear proteins.

The Expression Patterns of Tomato Swi3-Like Genes
In order to explore the possible roles of SlSWI3s, we first analyzed their tissue and organ specific expression profiles from publicly available RNA-seq datasets [32]. As shown in Figure 3A, the transcripts of all SlSWI3s were ubiquitously expressed in all tissues and organs. SlSWI3C and SlSWI3D had similar expression profiles and were expressed mainly in roots (R) and fruits from 1 cm to 10-day post breaker (B + 10) stages ( Figure 3A), suggesting that these genes may play redundant roles in root and fruit development. SlSWI3B showed a high expression in buds (B), flowers (F), roots (R) and B + 10 fruits, but its expression was low in other tissues. Compare with other SlSWI3s, the expression level of SlSWI3A was low in all tissues, especially in leaves (L) and B + 10 fruits ( Figure  3A). Indeed, all SlSWI3s displayed low expression patterns in L compared with other tissues ( Figure  3A).

The Expression Patterns of Tomato Swi3-Like Genes
In order to explore the possible roles of SlSWI3s, we first analyzed their tissue and organ specific expression profiles from publicly available RNA-seq datasets [32]. As shown in Figure 3A, the transcripts of all SlSWI3s were ubiquitously expressed in all tissues and organs. SlSWI3C and SlSWI3D had similar expression profiles and were expressed mainly in roots (R) and fruits from 1 cm to 10-day post breaker (B + 10) stages ( Figure 3A), suggesting that these genes may play redundant roles in root and fruit development. SlSWI3B showed a high expression in buds (B), flowers (F), roots (R) and B + 10 fruits, but its expression was low in other tissues. Compare with other SlSWI3s, the expression level of SlSWI3A was low in all tissues, especially in leaves (L) and B + 10 fruits ( Figure 3A). Indeed, all SlSWI3s displayed low expression patterns in L compared with other tissues ( Figure 3A).   Next, we investigated the expression pattern of SlSWI3s in response to environmental stimuli including hormones, salt, and cold by qRT-PCR. SlSWI3A and SlSWI3B were clearly induced by cold treatment, and SlSWI3B was also induced by ABA and salt treatments ( Figure 3B). The transcript of SlSWI3C was strongly repressed by ABA, salt and SA treatment. Unlike SlSWI3C, the expression of SlSWI3A and SlSWI3B was not changed under SA treatment ( Figure 3B). Interestingly, the transcript of SlSWI3D showed no difference under all treatments compared to control ( Figure 3B). These results revealed that SlSWI3A, SlSWI3B, and SlSWIC may be involved in response to different environmental stimuli in tomato.

Members of SlSWI3s Interact with Other Proteins
In Arabidopsis, yeast-two-hybrid screens showed that AtSWI3A and AtSWI3B interact with the MADS-box transcriptional factors AGL18 (AGAMOUS-LIKE 18) and AGL73 [33]. In addition, AtSWI3A and AtSWI3B form homodimers and heterodimers and interact with BSH/SNF5 [20], while AtSWI3B and AtSWI3C form heterodimers and interact with the Snf2-like protein BRM (the ATPase Next, we investigated the expression pattern of SlSWI3s in response to environmental stimuli including hormones, salt, and cold by qRT-PCR. SlSWI3A and SlSWI3B were clearly induced by cold treatment, and SlSWI3B was also induced by ABA and salt treatments ( Figure 3B). The transcript of SlSWI3C was strongly repressed by ABA, salt and SA treatment. Unlike SlSWI3C, the expression of SlSWI3A and SlSWI3B was not changed under SA treatment ( Figure 3B). Interestingly, the transcript of SlSWI3D showed no difference under all treatments compared to control ( Figure 3B). These results revealed that SlSWI3A, SlSWI3B, and SlSWIC may be involved in response to different environmental stimuli in tomato.

Members of SlSWI3s Interact with Other Proteins
In Arabidopsis, yeast-two-hybrid screens showed that AtSWI3A and AtSWI3B interact with the MADS-box transcriptional factors AGL18 (AGAMOUS-LIKE 18) and AGL73 [33]. In addition, AtSWI3A and AtSWI3B form homodimers and heterodimers and interact with BSH/SNF5 [20], while AtSWI3B and AtSWI3C form heterodimers and interact with the Snf2-like protein BRM (the ATPase of the SWI/SNF chromatin-remodeling complex) [18,34]. Thus, to explore whether SlSWI3s showed the similar interaction pattern, yeast-two-hybrid assays were performed. Like AtSWI3A and AtSWI3B, SlSWI3A, and SlSWI3B can also form heterodimers and homodimers ( Figure 4A, Figure S2). In addition, SlSWI3B also interacted with SlSWI3C in yeast cells ( Figure 4A). The interactions of SlSWI3s with the SlRIN and the Snf2-like protein SlCHR8 were also analyzed. Indeed, SlRIN is encoded by a member of the SEPALLATA4 (SEP4) clade of MADS-box genes [35], which function as a master regulator of the ripening process in tomato. SlCHR8 displays high sequence homology with BRM in Arabidopsis [36], and overexpression of SlCHR8 in tomato resulted in considerably compacter growth including significantly shorter roots and hypocotyls as well as reduced cotyledon and fruit size [37]. The yeast-two-hybrid assay showed that SlRIN interacted with SlSWI3A, SlSWI3B, and SlSWI3C, while SlCHR8 only interacted with SlSWI3B ( Figure 4A, Figure S2). Intriguingly, the yeast-two-hybrid assay also indicated that SlSWI3C can interacte with Arabidopsis SWI3A, SWI3B, and SWI3D, respectively ( Figure S3). Taken together, these results suggested that the function of SWI3s may be conserved in tomato and Arabidopsis. S2). In addition, SlSWI3B also interacted with SlSWI3C in yeast cells ( Figure 4A). The interactions of SlSWI3s with the SlRIN and the Snf2-like protein SlCHR8 were also analyzed. Indeed, SlRIN is encoded by a member of the SEPALLATA4 (SEP4) clade of MADS-box genes [35], which function as a master regulator of the ripening process in tomato. SlCHR8 displays high sequence homology with BRM in Arabidopsis [36], and overexpression of SlCHR8 in tomato resulted in considerably compacter growth including significantly shorter roots and hypocotyls as well as reduced cotyledon and fruit size [37]. The yeast-two-hybrid assay showed that SlRIN interacted with SlSWI3A, SlSWI3B, and SlSWI3C, while SlCHR8 only interacted with SlSWI3B ( Figure 4A, Figure S2). Intriguingly, the yeasttwo-hybrid assay also indicated that SlSWI3C can interacte with Arabidopsis SWI3A, SWI3B, and SWI3D, respectively ( Figure S3). Taken together, these results suggested that the function of SWI3s may be conserved in tomato and Arabidopsis. interacted with SlSWI3A, SlSWI3C SlRIN and SlCHR8 in yeast-two-hybrid assays. SlSWI3B was either cloned into pGBKT7 or pGADT7 vectors, whereas SlSWI3A, SlSWI3C and SlCHR8 were cloned into pGBKT7 vector, and SlRIN was cloned into pGADT7 vectors, respectively. Different constructs were cotransformed into the yeast strain AH109. The transformants were grown on the selective minimal medium without Leu and Trp (DDO). The transformants were also plated on QDO or QDO/X to test for possible interaction. QDO, SD/-Leu/-Trp/-His/-Ade. X, x-a-gal. The same results were obtained in three independent experiments, and one representative result was shown. (B) SlSWI3B interacted with SlSWI3A, SlSWI3C SlRIN and SlCHR8 by LCI assay. The A. tumefaciens carrying the indicated construct pairs were injected into tobacco leaves, and the luciferase activities were measured 2 d after injection. Similar results were obtained in three independent experiments, and one representative result was shown. Bars = 0.5 cm. (C) SlSWI3B interacted with SlRIN and SlCHR8 by Co-IP assay.The SlSWI3B and SlRIN (or SlCHR8) were subcloned into the pHB (Flag tag) and pEAQ-GFP (GFP tag) vector, respectively.These constructs were co-transformed into tobacco cells by Agrobacterium mediated infiltration. Transiently expressed SlSWI3B-Flag and SlRIN-GFP (or SlCHR8-GFP) were interacted with SlSWI3A, SlSWI3C SlRIN and SlCHR8 in yeast-two-hybrid assays. SlSWI3B was either cloned into pGBKT7 or pGADT7 vectors, whereas SlSWI3A, SlSWI3C and SlCHR8 were cloned into pGBKT7 vector, and SlRIN was cloned into pGADT7 vectors, respectively. Different constructs were cotransformed into the yeast strain AH109. The transformants were grown on the selective minimal medium without Leu and Trp (DDO). The transformants were also plated on QDO or QDO/X to test for possible interaction. QDO, SD/-Leu/-Trp/-His/-Ade. X, x-a-gal. The same results were obtained in three independent experiments, and one representative result was shown. (B) SlSWI3B interacted with SlSWI3A, SlSWI3C SlRIN and SlCHR8 by LCI assay. The A. tumefaciens carrying the indicated construct pairs were injected into tobacco leaves, and the luciferase activities were measured 2 d after injection. Similar results were obtained in three independent experiments, and one representative result was shown. Bars = 0.5 cm. (C) SlSWI3B interacted with SlRIN and SlCHR8 by Co-IP assay.The SlSWI3B and SlRIN (or SlCHR8) were subcloned into the pHB (Flag tag) and pEAQ-GFP (GFP tag) vector, respectively.These constructs were co-transformed into tobacco cells by Agrobacterium mediated infiltration. Transiently expressed SlSWI3B-Flag and SlRIN-GFP (or SlCHR8-GFP) were immunoprecipitated with an anti-GFP antibody, and then detected by western-blotting assay with an anti-Flag antibody.
To confirm their interaction in plant cells, SlSWI3A, SlSWI3B, SlSWI3C, SlRIN and SlCHR8 were fused with Nluc or Cluc and coexpressed in tobacco leaves and subjected for firefly luciferase complementation imaging assays. Consistent with the yeast-two-hybrid results, strong LUC activity was observed when Cluc-SlSWI3B and SlSWI3A-Nluc/or SlSWI3C-Nluc/or SlCHR8-Nluc were coexpressed ( Figure 4B). Similar results were also observed when SlSWI3B-Nluc and CLuc-SlRIN were coexpressed in tobacco leaves ( Figure 4B). Next, the interaction of SlSWI3B with SlRIN and SlCHR8 was examined by co-immunoprecipitation (Co-IP) assays. We transiently expressed SlSWI3B, SlRIN and SlCHR8 proteins in tobacco. The SlSWI3B fused with three FLAG tags (pHB-SlSWI3B) and SlRIN (or SlCHR8) fused with a GFP tag (pEAQ-SlRIN) constructs were co-transformed into tobacco epidermal cells by Agrobacterium-mediated infiltration assays. The anti-GFP antibody (GFP-Trap ® _A beads) was used for immunoprecipitation, and the immunoprecipitated protein was then analyzed by western-blotting assays using an anti-Flag antibody. We showed that SlSWI3B-FLAG protein was co-immunoprecipitated by SlRIN-GFP or SlCHR8-GFP ( Figure 4C). Collectively, these data supported that SlSWI3B interacts with SlSWI3A, SlSWI3C, SlCHR8, and SlRIN both in vivo and in vitro.

Ectopic Overexpression of SlSWI3C Enhances Leaf Growth
In Arabidopsis, overexpression of SWI3C frequently leads to an increase in rosette area [26]. To further analyze their functions, SlSWI3A, SlSWI3B, SlSWI3C, and SlSWI3D were ectopically expressed in Arabidopsis plants driven by the 35S promoter. The independent hygromycin-resistant T 1 transformants were transferred into soil and grown in a greenhouse and self-pollinated to obtain segregated T 2 progeny for genetic analysis. All overexpression lines showed a 3:1 segregation pattern for hygromycin resistance, and three homozygous lines were selected for further analysis. As for SlSWI3C, all these selected lines showed increased expression of SlSWI3C ( Figure S4). Phenotypic analysis showed that the 21-day-old soil-grown seedlings of 35S:SlSWI3C 1, 35S:SlSWI3C 2 and 35S:SlSWI3C 3 were much larger than Col-0 ( Figure 5A). Consistent with this observation, 35S:SlSWI3C 1, 35S:SlSWI3C 2 and 35S:SlSWI3C 3 plants had significantly larger rosette leaves ( Figure 5B). In addition, the surface area of the 5 th rosette leaf of 35S:SlSWI3C 1, 35S:SlSWI3C 2 and 35S:SlSWI3C 3 is 38.2, 46.04, and 46.08 mm 2 , respectively, which were significantly larger than that of Col-0 ( Figure 5C). Consistent with the larger leaves, the fresh weights (FW) of up-ground parts of all SlSWI3C overexpressing plants was increased when compared with Col-0 plants ( Figure S5). In contrast, transgenic Arabidopsis plants overexpression of SlSWI3A, SlSWI3B, and SlSWI3D showed no visible phenotypes including the rosette sizes compared with Col-0 ( Figure S4, Figure S6).   Next, we determined the role of SlSWI3C in the expression of the genes related to leaf development. As shown in Figure 5D, the expression levels of GROWTH REGULATING FACTOR 3 (GRF3), GRF5, and GRF6 were significantly increased in 21-day-old 35S:SlSWI3Cs shoots grown in long-day conditions compared with Col-0 ( Figure 5D). GRFs are comprised of nine members (GRF1 to GRF9) [38] and stimulate leaf cell proliferation, since overexpression of GRF1, GRF2, and GRF5 enhances leaf growth and cell division [39,40]. Thus, the increased expression of GRFs in 35S:SlSWI3Cs may be responsible for the larger leaf size. Intriguingly, HB33, a gene that is upregulated in brm mutants [26], was significantly downregulated in 35S:SlSWI3Cs plants ( Figure  5D).

Discussion
As one of the subunit of CRCs, SWI3 plays key roles in different cellular process in eukaryotic cells. In yeast, the SWI-SNF assembly and ATP-dependent H2A-H2B displacement are controlled by Swi3p [11,12]. Human BAF170 (SMARCC2) and BAF155 (SMARCC1), the homologs of Swi3p, are Next, we determined the role of SlSWI3C in the expression of the genes related to leaf development. As shown in Figure 5D, the expression levels of GROWTH REGULATING FACTOR 3 (GRF3), GRF5, and GRF6 were significantly increased in 21-day-old 35S:SlSWI3Cs shoots grown in long-day conditions compared with Col-0 ( Figure 5D). GRFs are comprised of nine members (GRF1 to GRF9) [38] and stimulate leaf cell proliferation, since overexpression of GRF1, GRF2, and GRF5 enhances leaf growth and cell division [39,40]. Thus, the increased expression of GRFs in 35S:SlSWI3Cs may be responsible for the larger leaf size. Intriguingly, HB33, a gene that is upregulated in brm mutants [26], was significantly downregulated in 35S:SlSWI3Cs plants ( Figure 5D).

Discussion
As one of the subunit of CRCs, SWI3 plays key roles in different cellular process in eukaryotic cells. In yeast, the SWI-SNF assembly and ATP-dependent H2A-H2B displacement are controlled by Swi3p [11,12]. Human BAF170 (SMARCC2) and BAF155 (SMARCC1), the homologs of Swi3p, are involved in cancers since BAF170 and BAF155 mutations were found in gastric and colorectal cancers and small cell lung cancers, respectively [41,42]. In Arabidopsis, both AtSWI3A and AtSWI3B are essential for early embryonic development, whereas AtSWI3C and AtSWI3D affect different phases of vegetative and reproductive development [20]. In this study, we identified four SWI3-like proteins in tomato: SWI3A, SWI3B, SWI3C, and SWI3D (Table 1, Figure 1A). Intriguingly, there are 6 SWI3-like proteins in rice (Table 1, Figure 1A) and 7 SWI3-like proteins in maize [27], indicating more SWI3-like proteins present in monocot. The fact that more SWI3 homologs exist in monocot may suggest the functional diversification of SWI3 paralogs in monocot.
All SlSWI3s contain the typical SWIRM and SANT domains ( Figure 1B). The SWIRM domain forms a compact helix-turn-helix (HTH)-related structure and mediates specific protein-protein interactions [15], while the SANT domain, which is characterized by its homology to the DNA binding domain of c-myb, functions as a histone tail binding module [17,43]. In addition to SWIRM and SANT domains, a SWIRM-assoc_1 domain was found in the C-terminus of SlSWI3s ( Figure 1B). A leucine zipper domain located on the C-terminus of SWI3s [20] is involved in the interaction between SWI3s, since human BAF155 and BAF170 form heterodimers (BAF155/BAF170) or homodimers (BAF155/155 or BAF170/170) through the leucine zipper domain [44]. The sequences of the SWIRM-assoc_1 domain are larger than that of leucine zipper domain and the function of the SWIRM-assoc_1 domain is still unclear. Similar to the Arabidopsis AtSWI3B [21], all SlSWI3s are also localized in the nucleus (Figure 2), which is consistent with their roles as the subunits of CRCs.
The tissue-specific and stress-responsive expression patterns may be a useful way to explore gene functions. The expression profiles of SlSWI3s suggested that some SlSWI3s may play a similar role with their homologs in Arabidopsis. For instance, SlSWI3C is mainly expressed in roots and reproductive organs ( Figure 3A), indicating that it functions in root and reproductive development [20]. Furthermore, functional divergence was also observed between SlSWI3B and its homolog AtSWI3B, since SlSWI3B is only poorly expressed in leaves ( Figure 3A), whereas AtSWI3B is highly expressed in this organ [45], and knock-down of AtSWI3B results in an upward-curling leaf phenotype [24]. In addition, we found that the expression of SlSWI3A and SlSWI3B is induced by cold treatment, while SlSWI3C is strongly repressed by ABA, salt and SA treatment ( Figure 3B). These data indicate that some SlCHRs may also respond to environmental stimuli.
In Arabidopsis, AtSWI3A and AtSWI3B can form homodimers and heterodimers, AtSWI3C can form heterodimers with both AtSWI3A and AtSWI3B, whereas AtSWI3D can form heterodimers only with AtSWI3B [18,20], suggesting their functional together in a complicated controlling network. In addition, AtSWI3B interacts with BSH/SNF5 and BRM, two core subunits of the SWI/SNF chromatin-remodeling complex [20,34]. Consistent with these data, our results also showed that SlSWI3A and SlSWI3B can form heterodimers and homodimers, whereas SlSWI3B and SlSWI3C can form heterodimers (Figure 4, Figure S2). Moreover, SlSWI3C can also form heterodimers with AtSWI3s ( Figure S3), and interacts with SlCHR8, the Arabidopsis homolog of BRM. Collectively, these data suggested that CRCs in Arabidopsis and tomato may have similar functions. Nevertheless, the interaction between SlSWI3s (SlSWI3A, SlSWI3B, and SlSWI3C) with the MADS-box protein SlRIN (Figure 4, Figure S2) indicated that SlSWI3s may be involved in fruit ripening in tomato.
Previous studies showed that AtSWI3B is essential for early embryonic development and ABA signaling [20,21], while AtSWI3C and AtSWI3D play important roles in vegetative and reproductive development [20] as well as GA signaling [25]. Mutations of AtSWI3C cause leaf curling and reduced fertility, while mutations of AtSWI3D lead to leaf curling, severe dwarfism, and alteration in the number and development of flower organs with complete male and female sterility [20]. Moreover, over-expression ofAtSWI3C increases the leaf size due to increased cell numbers [26]. AtSWI3B is also involved in leaf development via regulating IAMT1, which encodes a carboxyl methyltransferase in the auxin metabolism [24]. Intriguingly, the maize SWI3D-like gene ZmCHB101 knock-down lines also show curling leaves and impaired development in reproductive tissues, such as significant reduction of spikelet numbers and smaller and lighter ears compared to WT [27]. Collectively, these observations indicated that the physiological functions of SWI3s may be evolutionarily conserved in different plant species.
Like AtSWI3C, over-expression of SlSWI3C also resulted in the increased leaf size in transgenic Arabidopsis ( Figure 5A-C). The expression of GRF3, GRF5, and GRF6 was significantly increased in SlSWI3C overexpressing seedlings ( Figure 5D). These data suggested that the bigger leaf size of over-expression SlSWI3C transgenic Arabidopsis may be caused by the increased expression of GRFs, since overexpression of GRF1, GRF2, and GRF5 enhances leaf growth and cell division [39,40]. Nevertheless, transgenic Arabidopsis seedlings overexpression of SlSWI3A, SlSWI3B, and SlSWI3D showed no difference in the rosette sizes compared with Col-0 ( Figure S5), suggesting that SlSWI3C may function differently from other SISWI3 proteins. Taken together, our results indicated that the functions of SWI3C may, at least in some developmental processes, be conserved in tomato and Arabidospis. Further in-depth analysis using transgenic tomato is required to investigate the functions of SlSWI3s in tomato development.

Plant Materials and Growth Conditions
Solanum lycopersicum cultivar "Heinz 1706" was used in this study. Surface-sterilized tomato seeds were grown in the Murashige and Skoog (MS) medium with 1.5% sucrose and 0.8% agar for 14 days in a controlled environment greenhouse with a long photoperiod (16 h light/8 h dark) at 23 ± 1 • C.
For hormone and stress treatments, 14-day-old "Heinz 1706" seedlings grown in the MS medium were transferred to the liquid MS medium containing SA (5 mM), ABA (100 µM), and NaCl (200 mM) for 2 h, respectively. For cold stress test, the plates were transferred to a 4 • C growth cabinet for 2 h. After treatment, the seedlings were harvested and immediately frozen in liquid nitrogen for further gene expression. The seedlings without treatment (seedlings grown in the MS medium) were used as control. For each treatment, about 10 seedlings were used for RNA extraction.

Identification of Tomato Swi3-Like Genes and Phylogenetic Tree Construction
The AtSWI3A, AtSWI3B, AtSWI3C and AtSWI3D sequences of Arabidopsis thaliana and yeast ScSWI3 were used to perform a search in the Solamum lycopersicum genome using the BLASTP program in the SGN (http://solgenomics.net/). Then, the candidates of Swi3-like proteins were confirmed using the HMMER-based SMART (http://smart.embl-heidelberg.de/) and Pfam (http://pfam.xfam.org/) programs. The domain architecture was drawn using DOG2.0 software [46].

Subcellular Localization Assays
The subcellular localizations of SlSWI3s were first predicted using the WoLFPSORT (http://www. genscript.com/psort/wolf_psort.html), Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/#) and Euk-mPLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/euk-multi-2/) programs, and confirmed by GFP-tagged transient expression assays in tobacco (Nicotiana benthamiana) leaves [31]. The full length CDS of SlSWI3s were subcloned into the pEAQ-GFP vector (the C-terminus of SlSWI3s was fused with GFP) [48], and all the constructs were subsequently transformed into Agrobacterium tumefaciens strain GV3103. After grown in LB medium at 28 • C overnight, bacterial suspensions were infiltrated into young but fully expanded leaves of tobacco using a needleless syringe. After infiltration, plants were immediately covered with plastic bags and placed at 23 • C for 48 h before bag removal. The distribution of the fusion protein was determined using a confocal fluorescence microscope. To locate the fluorescent proteins in nuclei, the tobacco leaves were infiltrated with PBS containing 4 , 6-diamidino-2-phenylindole (DAPI, 1µg/mL). Three independent experiments were repeated to confirm results.

LCI Assay
Luciferase complementation imaging (LCI) assays were performed as described previously [49]. The CDS of SlSWI3A, SlSWI3B, SlSWI3C, SlCHR8, and SlRIN was cloned into either pCAMBIA-Nluc or pCAMBIA-Cluc. All the constructs were transformed into A. tumefaciens strain GV3103. An equal volume of A. tumefaciens harboring pCAMBIA-NLuc and pCAMBIA-CLuc (or their derivative constructs) was mixed to a final concentration of OD 600 = 1.0. Four different combinations of A. tumefaciens were infiltrated into four different positions at the same leaves of tobacco. Plants were placed in 23 • C and allowed to recover for 60 h. A low-light cooled CCD imaging apparatus (NightOWL II LB983 with indiGO software) was used to capture the LUC image. Three independent experiments were repeated to confirm results.

RNA Extraction and Expression Analyses
Total RNA was isolated using Trizol Reagent (Invitrogen, Shanghai, China) according to the manufacturer's protocol. cDNAs were synthesized from 2 µg of total RNA using the TransScript ™ One-Step gDNA Removal and cDNA Synthesis Supermix kit (TransGenBiotech, Guangzhou, China). Real-Time PCR was performed with iTaq ™ Universal SYBR ® Green Supermix (BIO-RAD, Shanghai, China) using ABI7500 Fast Real-Time PCR system. The gene-specific primers for real-time PCR were designed by PrimerQuest Tool (https://sg.idtdna.com/ Primerquest/Home/Index) and listed in Table S1. The Arabidopsis ACTIN2 (AT3G18780) and tomato ACTIN (Solyc03g078400) were used as a reference gene. Three independent sets of biological replicates were conducted with two technical replicates to confirm results.
For tissue and organ specific expression profiles, the expression data of tomato SlSWI3s were extracted from publicly available RNA-seq datasets from the Tomato Genome Consortium [32] and visualized with Matrix2PNG (https://matrix2png.msl.ubc.ca/bin/matrix2png.cgi) [51]. The publicly available RNA-seq data were obtained from transcriptome sequencing using three-week-old sand-grown seedlings, roots, leaves, buds (unopened flower buds), and flowers (fully open flowers) as well as fruits (at 1 cm, 2 cm, and 3 cm), MG (mature green), breaker (Br, early ripening), and 10-day post-breaker (B + 10, red ripe) stages of tomato "Heinz 1706" [32]. The expression data of SlSWI3s were normalized to have mean zero and variance one before producing the heat maps.

Generation of Transgenic Plants
To construct the SlSWI3C and SlSWI3D overexpression vectors, the CDS without a stop codon of SlSWI3C and SlSWI3D were cloned into the plasmid pHB-flag containing the 35S promoter [52]. The constructed plasmids were separately transformed into the A. tumefaciens strain GV3103 by the heat shock method. The bacteria carrying different constructs were used to transform WT (Col-0) plants via floral dip transformation.
Leaf areas were measured with ImageJ (http://rsb.info.nih.gov/ij/) using three-week-old seedlings grown in soil (the same stage seedlings were also for RNA isolation) after dissection of individual leaves. Rosette areas were calculated as the sum of the individual leaf areas. Three independent sets of biological replicates were conducted to confirm results.

Conclusions
In this study, four Swi3-like proteins, SlSWI3A, SlSWI3B, SlSWI3C, and SlSWI3D, were identified from tomato. All SlSWI3s contain two characteristic domains: SWIRM and SANT domains, and are localized in the nucleus. All SlSWI3s are ubiquitously expressed in all tissues and organs, and SlSWI3A and SlSWI3B can be induced by cold treatment. In addition, SlSWI3B forms homodimers with itself and heterodimers with SlSWI3A and SlSWI3C. SlSWI3B also interacts with SlRIN and SlCHR8, two proteins associated with tomato reproductive development, indicating that SlSWI3B may be involved in gene regulation in reproductive development. Furthermore, over-expression of SlSWI3C increases the leaf size in transgenic Arabidopsis with increased expression of GRF3, GRF5 and GRF6.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/20/ 5121/s1. Figure S1. The subcellular localization of tomato SWI3-like proteins. Figure S2. Self-activation (A) and the interactions of SlSWI3s with each other (B) were examined in yeast cells. Figure S3. The interactions of SlSWI3s with AtSWI3s were examined in yeast cells. Figure S4. The transcripts of SlSWI3s in 21-day-old transgenic Arabidopsis seedlings. Figure S5. The fresh weights (FW) of up-ground parts of Col-0 and SlSWI3C overexpressing plants grown in soil for two weeks. Figure S6. The phenotype of overexpression of SlSWI3A, SlSWI3B, and SlSWI3D transgenic Arabidopsis seedlings.