Genome-Wide Analysis of BURP Domain-Containing Gene Family in Solanum lycopersicum and Functional Analysis of SlRD1 Under Drought and Salt Stresses

Abstract

The BURP domain-containing (BURP) genes belong to plant-specific families and are known as essential for various biological processes in plants. However, knowledge of the functions of BURP genes in tomato (Solanum lycopersicum) is lacking. In our study, bioinformatics analysis was performed for the SlBURP gene family, including phylogeny, chromosomal localization, gene structure, cis-acting elements and expression. In addition, the function of SlRD1 in drought and salt stresses was explored. In tomato, fourteen BURP family members were identified, located on five chromosomes, including two tandem duplication clusters. These BURP members were classified into four subfamilies. The promoter regions of SlBURPs harbored numerous hormone- and stress-response elements. Tissue expression analysis showed that several SlBURPs were highly expressed in roots, flowers or fruits. Meanwhile, the expressions of most SlBURPs could be regulated by drought, salt and cold treatments, and some of them also responded to ABA treatment. Moreover, the ectopic expression of SlRD1 in Arabidopsis enhanced tolerances to drought and salt stresses and increased the sensitivity of seed germination to ABA. In conclusion, the comprehensive analysis of the SlBURP family in tomato and the functional exploration of SlRD1 in drought and salt stresses provide a basis for further dissecting the roles of tomato BURP genes.

1. Introduction

Tomato (Solanum lycopersicum L.) is a crucial vegetable crop and is cultivated worldwide [1]. However, various environmental stresses severely impact the yield and quality of tomato during its development [2]. Plants have evolved complex networks to respond to different environmental challenges. The BURP domain-containing (BURP) proteins have shown important functions in plant development and stress resistances [3,4,5]. Therefore, the study of tomato BURP genes could provide a theoretical basis for a better understanding of the stress response mechanism of tomato. The main feature of BURP proteins is the conserved BURP domain in the C-terminus, which included four repeated cysteine-histidine (CH) and several highly conserved amino acids. The BURP family’s name is based on the four protein members (BNM2, USP, RD22 and PG1β) previously discovered [6].

To date, the BURP gene family has been studied in numerous plants, including alfalfa (Medicago truncatula) [7], jujube (Ziziphus jujuba) [8], cotton (Gossypium hirsutum) [6], common bean (Phaseolus vulgaris) [9] and rose (Rosa chinensis) [10]. The BNM2-like, USP-like, RD22-like and PG1β-like subfamilies were four classic subfamilies of BURP family [10]. In addition, from further studies, other subfamilies were identified in the BURP family [6]. Studies on the functions of some BURP genes showed diverse roles during different processes of plant growth. For example, BNM2 (a BURP protein of Brassica napus), VfUSP (a BURP protein of Vicia faba) and ASG1 (a BURP protein of Panicum maximum) all might regulate spore formation during embryonic development [11,12,13]. OsRAFTIN1 and RA8 (two BURP proteins of rice) were involved in another development [14,15]. AtUSPL1, a member of the RD22-like subfamily, was involved in seed development [16]. Additionally, SCB1 (a BURP gene of soybean) was expressed specifically in the seed coat and might have an important role in the cell differentiation of the seed coat [17]. GhRD1, a BURP gene in cotton, activated by GhHOX3 and GhHD1, promoted cotton fiber elongation [18]. PG1β, a BURP protein in tomato, was involved in fruit maturation, as it affects pectin solubilization and degradation [19,20].

Moreover, many BURP genes, especially the members of the RD22-like subfamily, have been found to be involved in stress response. AtRD22 played a regulatory role in the drought stress response as an inhibitor and was also induced by abscisic acid (ABA) treatment [5]. MdRD22 could be activated by MdWRKY115 and enhances the resistance to drought and osmotic stress [21]. The overexpression of GmRD22 in Arabidopsis and rice could improve the tolerance to salt stress, possibly through increasing lignin production [22]. The expressions of BgBDC1, 2, 3 and 4, four BURP members of the RD22-like subfamily in the mangrove (Bruguiera gymnorrhiza), could be regulated by different abiotic stress and ABA treatments [23]. The overexpression of OsBURP16 could enhance the sensitivity of transgenic rice to various abiotic stresses through affecting pectin content of the cell wall [4]. The ectopic expression of PpBURP2 in rice could enhance the tolerance of plants to salt and drought stresses [3]. The ectopically expressed Sali3-2 in Arabidopsis seedings showed a higher tolerance to metal ions (cadmium and copper) [24]. These findings suggested that BURP genes, especially the member of the RD22-like subfamily, might also show important potential functions during tomato response to stress. Currently, the roles of BURP genes in many plants have been studied, with research focusing on plant development and stress responses. However, little has been reported about the functions of tomato BURP genes, especially in response to stress, which has limited our comprehension of tomato BURP family and resulted in a lack of knowledge on the roles of SlBURPs.

In order to better understand the potential roles of tomato BURP genes, a systematic analysis of BURP genes in tomato was performed, including gene structure, conserved motifs, phylogenic tree, chromosomal localization, cis-acting elements and expressions in various tomato tissues, under abiotic stress and ABA treatments. In the future, the tomato BURP genes could be chosen or used to deeply study according to the above results. Above all, the ectopic expression of SlRD1, a member of the RD22-like subfamily, enhanced the salt tolerance of transgenic Arabidopsis plants and improved ABA sensitivity of transgenic Arabidopsis seeds. The results demonstrated the function of SlRD1 in response to abiotic stresses and the association with ABA signaling pathway. These data are expected to provide a basis for further research on the functions of tomato BURP genes.

2. Results

2.1. Identification of BURP Gene in Tomato

The 14 BURP members (Table S1) in the tomato genome were identified via HMMER (Hidden Markov Model) screening with the BURP domain model file (PF03181), verified on the SMART website and named based on their homology with BURP members of Arabidopsis and other reported species. The length range of SlBURP proteins was from 132aa (SlUSP1) to 636aa (SlPG4), and their molecular weight ranged from 14.76 kDa to 69.47 kDa. The range of their isoelectric point values was from 5.13 to 9.71. The grand average of hydropathicity indicated that all SlBURP proteins, except SlPG5, were hydrophilic (Table S1).

2.2. Phylogenetic Analysis and Chromosomal Location of SlBURPs

In order to explore the phylogenetic relationships of SlBURP family members, 78 BURP proteins from tomato (14), Arabidopsis (5), rice (17), maize (10) and cotton (30) and two host BURP proteins (BNM2 and VfUSP) were used to construct the phylogenetic tree (Figure 1). The eight subfamilies, including four classic subfamilies, BURP V, BURP VI, BURP VII and BURP VIII were divided, which comprised BURP members from 1 (USP-like and BURP V subfamilies) to 5 (PG1β-like subfamily) species. Additionally, the PG1β-like and BURP VI subfamilies contained the most (19) and the fewest (3) BURP members, respectively. The BURP family members in tomato were distributed into BNM2-like, USP-like, RD22-like and PG1β-like subfamilies (Figure 1).

The distribution of SlBURPs on tomato chromosomes showed that these genes were unevenly located on five chromosomes (Figure 2). Among them, chromosome 05 contained the most SlBURPs (5), followed by chromosome 02 (4). Chromosomes 01 and 08 contained two SlBURPs, while chromosome 03 only contained one SlBURP (SlPG5). The analysis of duplication events in the SlBURP family showed two tandem duplication clusters, containing three (SlUSP1 to SlUSP3, and LePG1 to SlPG3) SlBURPs, respectively. The results indicated that the expansion of USP-like and PG1β-like subfamilies in tomato may derive from tandem duplication events.

2.3. Gene Structure and Conserved Motif Analysis of SlBURPs

The analyses of the intron exon structure and conserved motifs were performed to explore the conservation and diversification of tomato BURP family genes (Figure 3, Table S1). The results showed that six SlBURPs contained three exons and two introns, four SlBURPs contained two exons and one intron, three SlBURPs contained one exon, and SlRD1 contained four exons and three introns (Figure 3B and Table S1). The conserved domain analysis showed that all SlBURP proteins contained BURP domain, with half of them also containing a signal peptide (Figure S1A, Table S1). The conserved amino acid sites in the BURP domain were further analyzed using a multiple sequence alignment. The four repeat CH motifs in SlBURPs were summarized as CH-X₁₀-CH-X_25-27-CH-X₂₅-CH-X₈-W (Figure S1B).

In addition, the 10 conserved motifs in SlBURPs were further investigated (Figure 3C). The motifs in the same subfamily showed similar numbers and distributions. Among them, motif 2, motifs 1 and 3, and motif 4 were present in 14, 13 and 12 SlBURPs, respectively. Motifs 5, 8, 9 and 10 were only present in the PG1β-like subfamily. Motif 6 was present in the USP-like and PG1β-like subfamilies. Motif 7 was present in the USP-like and RD22-like subfamilies. Overall, these results revealed that closely related SlBURPs had more similar gene structures and motif arrangements.

2.4. Cis-Acting Element Analysis in Putative SlBURP Promoters

We analyzed the cis-acting elements in the promoters of SlBURPs to explore the potential roles of SlBURPs (Figure 4). Four types of elements, including hormone response, stress response, plant development and transcription factor binding elements, were investigated. The hormone response elements included response to ABA (AREB), ethylene (ERE), auxin (AuxRR-core and TGA-box), gibberellin (GARE, P-box and TATC-box), methyl jasmonate (CGTCA-motif and TGACG-motif) and salicylic acid (TCA-element) elements. Notably, ABA- and ethylene-responsive elements were widely distributed in SlBURP promoters, with 11 and 9, respectively. The stress response elements, especially MYC, WUN-motif and ARE, were ubiquitously present in all SlBURP promoters. The plant development elements were present in 10 SlBURP promoters. Among them, the AAGAA-motif, involved in secondary xylem development, was most widespread, being present in seven SlBURP promoters. In addition, binding sites for the WRKY and MYB transcription factors were present in several SlBURP promoters, indicating that these SlBURPs may be regulated by upstream transcription factors. These results indicated that SlBURPs may be involved in abiotic stress response and hormone response.

2.5. Tissue Expression of SlBURPs

To explore the potential functions of SlBURPs during tomato growth, qRT-PCR was used to analyze the expression of 10 SlBURPs (excluding SlUSP1-SlUSP3 for high sequence similarity and SlPG4 for almost no expression in different tissues) in various tomato tissues, including roots, stems, leaves, flowers and fruits at different stages (Figure 5). The expressions of SlBURPs showed spatio-temporal specificity. SlPG2, SlBNM1, SlBNM2 and SlPG5 were highly expressed in different tissues, except fruits, at different stages. Notably, LePG1 and SlRD1 showed generally high expression in different tissues, especially in breaker stage or red fruit. Additionally, SlUSP5 and SlUSP6 exhibited root specific expressions, and SlUSP4 and SlPG3 were highly expressed in flowers. The results indicate that SlBURPs may perform various functions in plant development.

2.6. Expression Analysis of SlBURPs Under Abiotic Treatments

The stress-responsive elements detected in SlBURP promoters indicated that SlBURPs may be related to tomato’s response to abiotic stress (Figure 4). Therefore, we explored the expression dynamics of SlBURPs (except SlUSP4 and SlUSP5 with undetectable expressions in leaves both under control and treatments) under drought (20% PEG-6000), salt (200 mM and 400 mM) and cold (4 °C) stresses. As shown in Figure 6, the expressions of all eight SlBURPs were induced by the drought, salt and cold treatments. Under drought treatment, the expression levels of SlBNM2 and SlUSP6 were significantly down-regulated. In contrast, the expression levels of SlPG2 and SlPG5 were significantly up-regulated. The remaining SlBURPs showed alternately induced expression dynamics (Figure 6A). Under salt treatment, the expressions of SlRD1, SlBNM1, SlUSP6 and SlPG3 were significantly up-regulated at 400 mM or both 200 mM and 400 mM conditions, with SlRD1 showing the most significant up-regulation (5.72-fold). The expression of LePG1 was significantly down-regulated at 3 days of treatment. The expressions of SlBNM2, SlPG2 and SlPG5 were not significantly changed under 200 mM salt treatment but were significantly up-regulated at 12 h and 24 h, and then significantly down-regulated at 3 days under 400 mM salt treatment (Figure 6B). Under cold treatment, the expressions of SlPG3 and SlUSP6 were significantly up-regulated, while those of SlBNM1 and LePG1 were significantly down-regulated. The expressions of the remaining SlBURPs were significantly up-regulated over 6–24 h or at 24 h, and then significantly down-regulated at 48 h (Figure 6C). Overall, the variable induction of SlBURPs under the tree treatments indicated their potential roles in response to abiotic stresses.

2.7. Expression Analysis of SlBURPs Under ABA Treatment

The expression of eight SlBURPs were investigated by qRT-PCR to explore whether SlBURPs were responsive to ABA treatment. The result showed that the expressions of 5 SlBURPs (except SlBNM2, SlUSP6 and SlPG3) were sensitive to ABA treatment (Figure 7). Among them, SlRD1, SlBNM1 and SlPG5 showed significantly up-regulated expressions, with constant increasing in the expression of SlRD1 and most significantly up-regulated at 12 h (10.82-fold) in the expression of SlBNM1. The expression of LePG1 were significantly down-regulated at 3 h. The expression of SlPG2 were significantly up-regulated at 3 h, then down-regulated at 6 h. The result suggested certain SlBURPs may be involved in ABA response pathway.

2.8. SlRD1 Improved Salt and Drought Tolerance of Transgenic Arabidopsis

Based on the phylogenetic analysis (Figure 1), we found that SlRD1 was most homologous with AtRD22, a drought-responsive BURP gene [5]. Furthermore, cis-acting element analysis (Figure 4) and expression analysis under abiotic stress and ABA treatments (Figure 6 and Figure 7) showed that stress- and ABA-responsive elements were present in the SlRD1 promoter, and the expression of SlRD1 was significantly induced by the three abiotic stress and ABA treatments, especially salt and ABA. To further explore the potential function of SlRD1, we employed the ectopic expression of SlRD1 in Arabidopsis. Three successful transformations of SlRD1 into Arabidopsis lines (OE#3, OE#6 and OE#8) were verified using qRT-PCR (Figure S2A). The primary roots of 5-day-old transgenic Arabidopsis seedlings grown on 1/2 Murashige and Skoog (MS) media were significantly longer than those of wild-type (WT) Arabidopsis (Figure S2B,C). Furthermore, after 5 days of 100 mM mannitol and 100 mM NaCl treatments, respectively, the yellowing and wilting of transgenic Arabidopsis leaves were less severe compared with the WT leaves (Figure 8A). The survival rates of transgenic Arabidopsis plants under drought and salt treatments were both significantly higher than those of the WT (Figure 8B). The primary roots of transgenic Arabidopsis plants were significantly longer compared with the WT under the control, drought and salt treatments (Figure 8C). In addition, after 200 mM NaCl treatment (7 days) and drought treatment (3 days and 10 days), transgenic Arabidopsis plants showed higher resistances than the WT (Figures S3 and S4). These results indicated that overexpression of SlRD1 improved drought and salt tolerance in Arabidopsis, potentially through promoting root elongation.

2.9. SlRD1 Improved ABA Sensitivity of Transgenic Arabidopsis Seeds

The expression of SlRD1 was significantly and constantly up-regulated under ABA treatment (Figure 7). In order to investigate whether SlRD1 was involved in ABA response, the germination rates of transgenic Arabidopsis lines and the WT under control and ABA treatments were counted (Figure 9). The germination rates showed no significant difference between transgenic Arabidopsis seeds and WT grown on 1/2 MS media from 0 day to 7 days (Figure 9B). However, the germination rates of transgenic Arabidopsis seeds and WT were both reduced when grown on 1/2 MS media with 10 μM ABA. Notably, the germination rates of transgenic Arabidopsis seeds were significantly lower than the WT from 3 days to 6 days (Figure 9C). The results indicated that the ectopic expression of SlRD1 in Arabidopsis enhanced sensitivity to exogenous ABA.

3. Discussion

Many BURP genes have been studied for their critical roles in various developmental processes and stress responses [3,5,18,21]. With the broadening of plant genomics publications and research, the genome-wide identification and analysis of BURP gene family also have been studied in many plants. In our study, the phylogenetic analysis of BURP members revealed that eight subfamilies were classified, with fourteen tomato BURP members distributed in BNM2-like, USP-like, RD22-like and PG1β-like subfamilies (Figure 1). The similarity of gene structure and conserved motif arrangement of SlBURPs within same subfamily supported the classification of subfamilies. The various numbers of exons and motifs of SlBURPs from different subfamilies may be related to the functional diversity. The similar results of gene structures were found in cotton [6], rose [10] and legumes [25]. Also, the phylogenetic results in BURPs of cotton [6], soybean [26] and alfalfa (Medicago truncatula) [7] were found. Gene duplication events and uneven distribution of chromosomes (Figure 2) have been observed in the tomato BURP family indicating that this family may have an expansion and functional redundancy, similar to findings in cotton [6] and alfalfa [7].

The roles of BURP family genes in plant-specific developmental processes, including seeds [17], fruit [19,20] and flowers [14,15], have been widely reported. The expression patterns of SlBURPs in different tomato tissues were investigated to assess the potential functions in tomato development (Figure 5). The expressions of some SlBURPs showed distinct tissue-specificity. For instance, SlBNM1 and SlBNM2 were more prevalent in roots than other tissues, similar to the homologous AtUSP1 [5]. SlUSP5 and SlUSP6 showed root-specific expressions, indicating the potential roles of these genes in root development. The fruit specific expression (especially in tomato fruit ripening) of LePG1 has been studied [20] and were also proven in our study. Although belonging to the PG1β-like subfamily, SlPG3 showed a similar expression pattern to LePG1, while SlPG2 and SlPG5 exhibited different expression patterns, with high expression in the stem. Similarly, the conservation and divergence of BURP expressions have also been found in jujube [8] and soybean [26]. In addition, SlRD1 exhibited high expression in leaf, similar to AtRD1 [5], and also has the highest expression in fruit, suggesting a variety of biological functions during plant evolution.

The stress response elements were widely present in SlBURP promoters (Figure 4), similar to previous studies [6,7,8], indicating the possible response to various stresses. The result of qRT-PCR analysis showed that the expressions of all SlBURPs could be induced by drought, salt, and cold treatments, although with different levels of induction and trends (Figure 6). Under drought treatment, the expression of SlPG3 showed the most significant up-regulation at 24 h, followed by SlPG2 at 24 h and SlRD1 at 1 h, which were similar to PvBURP4/8, MtBURP30/31/32 (belonging to PG1β-like subfamilies), PvBURP3 and MtBURP10/11 (belonging to RD22-like subfamilies) expression in Phaseolus vulgaris [9] and alfalfa [7]. In addition, SlUSP6 showed strikingly down-regulated expression, indicating this gene may have a negative response to drought stress. Under salt treatment, the expressions of most SlBURPs, except LePG1, were up-regulated at 12 h and 24 h, similarly to the expressions of ZjBURPs and GhBURPs under salt stress [6,8]. Under cold treatment, the expressions of most SlBURPs, except LePG1 and SlBNM1, were significantly up-regulated at 24 h, which was consistent with expressions of CaBURP in Cicer arietinum [25], whereas the opposite was shown for ZjBURPs [8]. This indicated that BURP genes of different species may play various roles in plant response to cold. ABA showed an important role in plant response to abiotic stress [27]. Five SlBURPs, specifically SlRD1 and SlBNM1 with the most significantly up-regulated expressions at different points, showed various responses to ABA treatment (Figure 7) and similar expressions were observed for the PvBURP family [9] and ZmRD22B [28].

The identification and expressions of BURP genes have been performed in many plants, but their largely unknown functions provide limited understanding of this gene family. Based on the homologous stress-related AtRD22 and its high response to multiple abiotic stresses and ABA treatment, SlRD1 was chosen to be ectopically expressed in Arabidopsis to further explore the role of this gene in response to stress. Higher resistance was detected in transgenic Arabidopsis plants than WT indicating that SlRD1 may positively regulate the response to drought and salt stresses (Figure 8, Figures S3 and S4). In Arabidopsis, AtRD22 was involved in the drought stress response [5]. Ectopic expression of BnBDC1, homologous to AtRD22, enhanced the drought and cold resistance of Arabidopsis [29]. Similarly, ectopic expression of GmRD22 improved the resistance of Arabidopsis and rice to salt stress [22]. The ectopic expression of RvBURP4 in Arabidopsis suggested the diverse functions of this gene in response to salt and drought stresses [10]. These results suggest that BURPs, especially members of the RD22-like subfamily, played critical roles in the plant’s abiotic stress response. In addition, the higher sensitivity of transgenic Arabidopsis seeds to ABA treatment than those of the WT indicated that SlRD1 may be involved in the ABA signaling pathway (Figure 9). This result was supported by the four ABA response elements (AREB) in SlRD1 promoter (Figure 4) and the significantly up-regulated expression of SlRD1 under ABA treatment at all treatment points (Figure 7). AtRD22 was part of the ABA-mediated drought stress response [5]. This is consistent with another study that reported that the RcBURP4 transgenic Arabidopsis was more susceptible to ABA treatment than the WT during seed germination [10].

4. Materials and Methods

4.1. SlBURP Family Member Identification in Tomato

The genome sequences of tomato (version ITAG4.0) and BURP domain model file (PF03181) were obtained from Sol Genomics Network (http://solgenomics.net/ (accessed on 19 September 2023)) and the Pfam database (https://pfam.xfam.org/ (accessed on 19 September 2023) [30], respectively. The candidate SlBURP genes from tomato genome were screened using HMMER 3.0 with the threshold set as E-value < 1 × 10⁻¹⁰ [6]. The NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 19 September 2023)) and SMART (http://smart.embl-heidelberg.de/ (accessed on 19 September 2023)) were used to individually identify the BURP domain in SlBURP protein sequences [31]. The information of SlBURP proteins, including molecular weights (Mw), isoelectric points (pI) and grand average of hydropathicity, was predicted using the ExPASy online tool (http://web.expasy.org/protparam/ (accessed on 19 September 2023)) [32].

4.2. SlBURP Phylogeny, Chromosomal Location and Duplication Analysis

The BURP protein sequences of Arabidopsis, rice, maize, cotton and two host BURP proteins (BNM2 and VfUSP) were downloaded from TAIR (https://www.arabidopsis.org/ (accessed on 22 September 2023)), Phytozome 13 (https://phytozome-next.jgi.doe.gov/ (accessed on 22 September 2023)) and NCBI ((www.ncbi.nlm.nih.gov/protein (accessed on 22 September 2023)). The phylogenetic trees were established using the Neighbor-Joining method in MEGA 7.0, and the bootstrap replications was set to 1000 [33].

The chromosomal location of SlBURPs was generated using TBtools, based on the genomic location information from the gff3 file of the tomato genome [34]. Multiple sequence alignment of SlBURPs was performed using Clustal Omega on the EMBL-EBI website (https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 9 January 2024)). The segmental duplication genes were confirmed if the alignment coverage and the sequence similarities were ≥80%. Moreover, if the physical locations of the duplicated genes on the same chromosome were ≤200 kb apart, these genes were regarded as tandem duplication clusters [35].

4.3. SlBURP Gene Structures and Conserved Motifs

The gene structure image of SlBURPs was drawn using GSDS 2.0 (http://gsds.cbi.pku.edu.cn/ (accessed on 10 January 2024), based on the intron-exon position from the gff3 file of the tomato genome. The conserved BURP domain and amino acid sites of SlBURP proteins were analyzed using DNAMAN 8.0. SignalP 4.0 Server (http://www.cbs.dtu.dk/services/SignalP/ (accessed on 10 January 2024)) was employed to predicate the signal peptides in SlBURP proteins [36]. The conserved motifs of SlBURP proteins were predicted using the MEME online tool (https://meme-suite.org/meme/tools/meme (accessed on 10 January 2024)) with 10 motifs as maximum number.

4.4. Cis-Acting Elements in SlBURP Promoters

The 1500 bp upstream sequences from the start codon of SlBURPs were extracted from the tomato genome via TBtools [34]. Then, the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 21 September 2023)) was employed to investigated potential cis-acting elements in SlBURP promoters [37].

4.5. Plant Growth and Treatments

The tomato cv. ‘Micro-Tom’ was sown and transplanted in a growth chamber under light conditions of 26 °C for 16 h and dark conditions of 18 °C for 8 h. The different tomato tissues were collected and frozen in liquid nitrogen, and then stored at −80 °C. The six-leaf-stage tomato seedlings were used for the abiotic stress and ABA treatments. For drought and salt treatments, the tomato seedlings were irrigated with 20% (w/v) PEG6000 or 200 mM NaCl or 400 mM NaCl. The leaf samples were collected at 0, 1, 3, 6, 12, and 24 h for PEG6000 treatment and 0, 12, 24 h and 3 days for NaCl treatment. For cold treatment, the tomato seedlings were transferred to an artificial climate chamber set at 4 °C. The leaf samples were collected at 0, 6, 12, 24, and 48 h. For ABA treatment, the leaves of the tomato seedlings were sprayed with 100 μM ABA and collected at 0, 3, 6, 12, 24, and 48 h. Each time point of treatment contained tree biological replicates with five tomato seedlings. The collected leaf samples were rapidly frozen in liquid nitrogen and stored at −80 °C for further analysis.

4.6. RNA Extraction and qRT-PCR Analysis

The Polysaccharide Polyphenol Plant RNA Extraction kit (Tiangen, Beijing, China) and StarScript II First-strand cDNA Synthesis Mix with gDNA Remover (GenStar, Beijing, China) were chosen for RNA extraction and cDNA synthesis, respectively. RealStar Green Fast Mixture with ROX (2×) (GenStar, Beijing, China) was used for qRT-PCR via an Applied Biosystems StepOnePlus instrument. The relative expressions of SlBURPs were calculated using the 2^−ΔΔCT method. And, SlEF1α and Actin were used as the internal reference genes in tomato and Arabidopsis, respectively. Then, the t-test was used to analyze the significance of differences in SlBURP expressions. The specific primers of SlBURPs were designed using Primer 5.0 and were listed in Table S2.

4.7. Generation of SlRD1 Transgenic Arabidopsis Plants

The CDS of SlRD1 was amplificated, and then the correct sequence was cloned into the overexpression vector (pBI121). The recombinant plasmid, 35S::SlRD1, was obtained and transformed into an Agrobacterium strain (GV3101). The floral dripping method was used to transform the Arabidopsis cv. ‘Col-0’ [38]. The T₀ transgenic Arabidopsis seeds were screened on 1/2 MS media with 15 µg/L. The SlRD1 expressions of T₃ homozygous transgenic Arabidopsis seedings were detected using qRT-PCR. The primers used were listed in Table S2.

4.8. Stress Treatments of SlRD1 Transgenic Arabidopsis Plants

The WT and transgenic Arabidopsis seeds were evenly sown on 1/2 MS media and grown normally for 5 days in a plant growth chamber under light conditions at 22 °C for 16 h and dark for 8 h. The lengths of primary roots were measured and statistically analyzed with three biological replicates. For the stress treatment assays, 3-day-old WT and transgenic Arabidopsis seedings were transferred to 1/2 MS, 1/2 MS with 100 mM mannitol and 100 mM NaCl media, respectively. The lengths of the primary roots and survival rates were calculated and statistically analyzed after 5 days under normal, drought and salt conditions with three biological replicates. Four-week-old WT and transgenic Arabidopsis seedings grown in soil were chosen to 200 mM NaCl and drought treatments, respectively.

For the Arabidopsis seed germination assays, the WT and transgenic Arabidopsis seeds were sterilized and then evenly sown on 1/2 MS and 1/2 MS with 10 µM ABA media, respectively. Then, the seed germination rates were calculated and statistically analyzed with three biological replicates from days 0 to 7.

5. Conclusions

A total of fourteen SlBURP genes were confirmed in the tomato genome and classified into BNM2-like, USP-like, RD22-like and PG1β-like subfamilies. The closely related SlBURPs contained similar gene structures and motif arrangements. Tandem duplication may be the main cause of tomato BURP gene family expansion. The expressions of SlBURPs in different tomato tissues were revealed to be spatio-temporally specific. The analyses of cis-acting elements and expression dynamics under abiotic stress and ABA treatments implied that SlBURPs may play important roles in response to various stresses. Moreover, the ectopic expression of SlRD1 in Arabidopsis facilitated the elongation of primary roots under normal, drought and salt conditions. The tolerance of SlRD1 transgenic Arabidopsis under drought and salt treatments was higher compared with the WT, suggesting a positive role for this gene in response to drought and salt stresses. In summary, our study deepens the understanding of the BURP gene family and provides a foundation for further exploration of the potential functions of tomato BURP genes.