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Article

Genome-Wide Identification, Cloning and Expression Profile of RanBP2-Type Zinc Finger Protein Genes in Tomato

by
Yanna Gao
1,†,
Ning Li
1,†,
Jiaojiao Ruan
1,
Ying Li
1,
Xiaoli Liao
2 and
Changxian Yang
2,*
1
College of Horticulture, Henan Agricultural University, Zhengzhou 450002, China
2
Key Laboratory of Horticultural Plant Biology (MOE), Huazhong Agricultural University, Wuhan 430070, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2022, 8(11), 985; https://doi.org/10.3390/horticulturae8110985
Submission received: 19 September 2022 / Revised: 20 October 2022 / Accepted: 20 October 2022 / Published: 23 October 2022
(This article belongs to the Special Issue Genes, Genetics and Breeding of Tomato)
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Abstract

:
The RanBP2-type zinc finger (RBZ) protein genes, which are well-characterized in animals, are involved in the regulation of mRNA processing. Although they are diversely distributed in plants, their functions still remain largely unknown. In this study, we performed a comprehensive bioinformatic analysis of 22 RBZ genes in tomato. The gene structure analysis revealed that the SlRBZ genes have 2 to 17 exons. SlRBZ proteins contain typical conserved domains, including Motif 1 or Motif 2, or a combination of Motif 9 and Motif 4. Two paralogous pairs were identified in the tomato. Segmental duplication possibly contributed to the expansion of the SlRBZ genes in tomato. Interestingly, the SlRBZ15 gene generated four products, yielded by alternative splicing. A cis-regulatory element analysis revealed that SlRBZ genes might be involved in the complex regulatory networks during plant growth and development. The expression profiles of the SlRBZ genes were analyzed in different tissues using eight phytohormones and four abiotic stress treatments based on RNA sequencing data and qRT-PCR verification. The results showed that each gene responded differently to more than one phytohormone or abiotic stress type. This research provides a foundation for future functional research on SlRBZ genes in tomato.

1. Introduction

The zinc finger protein superfamily comprises several subfamilies that are characterized by various structural and functional differences. Earlier studies showed that zinc finger proteins participate in a variety of biological processes, such as plant growth and development, as well as the plant’s adaptation to stress and hormone responses [1]. The zinc finger (Znf) domain, which has been identified in eukaryotes, is the most well-known DNA binding domain in numerous transcription factors. One zinc finger family without the classic DNA binding function is the RBZ family, named after Ran Binding Protein 2 in humans [2]. The RanBP2 Znf domain (PFAM00641, IPR001876), with the conserved sequence pattern W-X-C-X(2,4)-C-X(3)-N-X(6)-C-X(2)-C, represents a new superfamily of C2C2-type zinc finger motifs. Although most members of this superfamily contain one copy of the RanBP2 Znf domain, some consist of several RanBP2 Znf domains [3].
Several human RBZ proteins, including ZRANB2, EWS, TLS/FUS, RBP56, RBM5, RBM10, and TEX13A, can bind to the cis-element GGU that is usually involved in regulation of mRNA processing in humans [4]. Previous studies have reported that RBZ genes participate in the differentiation and development of several organs and tissues, including seed maturation, follower development and chlorophyll biosynthesis in plants [5,6,7,8]. For example, the GhRBZ gene, expressed in different developmental stages of cotton glands, may play an important role in the development of the cotton gland [9]. A total of 24 RBZ genes have been identified in Arabidopsis thaliana [10]. TATA-binding-protein-associated factor 15b suppresses flowering by repressing the transcription elongation of flowering locus C [5]. Histone Deacetylase 15 (HDA15) represses chlorophyll biosynthesis in the dark, mediated by PIF3 [7]. The suppressor of ABSCISIC ACID-INSENSITIVE (ABI)3–5 regulates seed maturation by preventing the splicing of introns in ABI3 [8]. There are four organelle-zinc-finger (OZ)-editing factors in Arabidopsis, including OZ1 (two RanBP2 Znf domains), OZ2 (two RanBP2 Znf domains), OZ3 (three RanBP2 Znf domains), and OZ4 (four RanBP2 Znf domains). Two of the OZ proteins function as key proteins in RNA processing in their respective organelles, and the remaining two are present in plastids or mitochondria [11]. OZ1, a key chloroplast RNA-editing factor, plays an important role in chloroplast development and germination rates in Arabidopsis [11]. The oz2 null mutant presents an embryo-lethal phenotype [12]. The null mutant can be rescued by expressing the wild-type OZ2 under the control of the seed-specific ABI3 promoter. Recent results showed that OZ2 is a mitochondrial splicing factor rather than an editing factor [13].
Several RBZ genes are reportedly involved in signaling pathways and biotic stimuli, including ABA, gibberellin (GA), and heat shock in plants [6,14,15,16]. MYB96 induces the expression of HDA15 and, subsequently, inhibits the expression of abscisic-acid (ABA)-signaling genes [15]. HDA15 interacts with HFR1 to repress warm-temperature responses [16]. Stress-associated RNA binding protein 1, containing three RanBP2 Znf domains without other known motifs, acts as a post-transcriptional regulatory protein. Stress-associated RNA binding protein 1 regulates the ABA signaling pathway by binding to the 3′ UTR AUUUA sequences of ABI2. In addition, it has been determined that rhomboid-like (RBL) protein 10 may be upregulated in response to heat shock [14].
Tomato (Solanum lycopersicum) is an economically important horticultural crop worldwide due to its good flavor and high nutrient value. Moreover, it is a fleshy fruit and model plant that is suitable for genetic and genomic studies. Research on the gene functions and breeding of tomato has accelerated following the sequencing, resequencing and pan-genomic analysis of wild and cultivated tomatoes [17,18,19,20]. To date, only one SlRBZ gene (SlRBZ6) in tomato has been characterized. SlRBZ6 is a constitutive gene with four RanBP2 Znf domains, which may inhibit the biosynthesis of chlorophyll, carotenoids and gibberellin by blocking chloroplast development [6]. However, the characteristics and biological functions of the other majority of SlRBZ genes in tomato remain unknown.
In this study, we performed the genomic-wide identification and bioinformatic analysis of the SlRBZ gene family of tomato. A total of 22 SlRBZ genes were identified and isolated from the tomato. The gene structures, replication events, phylogeny, conserved motifs and cis-regulatory elements of the promoters of all the putative SlRBZ genes were analyzed. Moreover, we investigated the expression profiles of the genes only possessing RanBP2 Znf domains in different tissues using eight phytohormones and four abiotic stresses treatments by real-time quantitative PCR (qRT-PCR). The study provides a useful reference for the functional characterization of SlRBZ members in the tomato developmental processes.

2. Materials and Methods

2.1. Plant Growth Conditions and Hormone and Stress Treatments

Solanum lycopersicum L. ‘Ailsa Craig’ tomatoes were used as the research materials. The seeds were germinated and grown in a greenhouse (120 μmol photons/m2s, 16/8 h light/dark regimen, 25 ± 2 °C, and 60% relative humidity). The transcription levels of SlRBZ genes in one-month-old seedlings were analyzed after being subjected to several abiotic stresses and plant hormone treatments. For the phytohormone treatments, the seedlings were sprayed with solutions containing 100 µM salicylic acid (SA), 100 µM indole-3-acetic acid (IAA), 100 µM ABA, 100 µM 4-(indolyl)-butyric acid (IBA), 100 µM 6-benzylaminopurine (6-BA), 100 µM jasmonic acid (JA), 100 µM Gibberellin A3 (GA) and 1% (v/v) ethephon (ETH). A mock treatment was sprayed with water as the control. Seedling shoots were collected after 0.25, 0.5, 1, 3, 6, 9, 12, 24 and 36 h of treatment. Cold, heat, and salt stresses were applied, as we previously described [21]. Drought stress was applied using the method described in Lu et al. (2012) [22]. The samples were collected after 0.5, 1, 3, 6, 9, 12 and 24 h of treatment. Different tissues from adult plants were harvested as we described previously [21]. All collected samples were frozen in liquid-N2 and stored at −80 °C in an ultralow-temperature refrigerator until further use.

2.2. The qRT-PCR

Total RNA was extracted using TRIzol reagent (Invitrogen, Waltham, MA, USA). The genome DNA was removed with DNase I (Takara). The cDNA was synthesized using the Transcriptor High-Fidelity cDNA Synthesis Kit (Roche). Primer 3.0 was used to design primers (Table S1). The experiment was conducted using the following parameters: a melting temperature of 60–62 °C, primer length of 23 ± 2 bp, GC content of 40–60% and product size of 80–200 bp. Actin was used as an internal reference for normalization. The qRT-PCR was performed using an optical 96-well plate with a LightCycler 480 system (Roche Diagnostics, Basel, Switzerland), according to the method described by Gao et al. (2015) [21]. The 2−△△Ct method was used to calculate the relative expression levels of these genes [23].

2.3. Identification and Cloning of SlRBZ Genes

The consensus pattern of the RanBP2 Znf domain (PFAM00641, IPR001876) is W-X-C-X (2, 4)-C-X (3)-N-X (6)-C-X (2)-C (3). The HMMER 3.0 program was employed to search for proteins with a RanBP2 Znf domain in the tomato database. The conserved domain BLASTP searches were also performed using the tomato amino acid sequences of SlRBZ6 in the SOL Genomics Network (E < 10−5). To confirm the results, protein sequences of the entire candidate genes were further analyzed for the presence of RanBP2 Znf domains using the online Conserved Domain Database of NCBI (https://www.ncbi.nlm.nih.gov/cdd, accessed on 20 October 2022) and SMART (http://smart.embl-heidelberg.de/, accessed on 20 October 2022). The nucleotide and amino acid sequences of the predicted SlRBZ genes were downloaded (File S1). ProtParam (https://web.expasy.org/protparam/, accessed on 20 October 2022) was used to calculate the basic physical and chemical characteristics (theoretical isoelectric point (PI), molecular weight (MW), etc.) of the tomato SlRBZ proteins. Primers (Table S1) were designed using Primer 5.0 version. Coding sequences of the SlRBZ genes from the tomato cultivar Ailsa Craig were amplified by PCR using Phanta® Super-Fidelity DNA Polymerase. The PCR products were purified and ligated to a pEasy-Blunt vector (TransGen Biotech, Beijing, China) for sequencing.

2.4. Gene Structure, Conserved Motifs and Cis-Regulatory Elements of SlRBZ Genes

To predict the exon–intron structure of the SlRBZ genes, a comparison between the genomic sequences and their full-length cDNA sequences was performed using the GSDS 2.0 website [24]. MEME online software was used to investigate the conserved motifs encoded by each SlRBZ gene [25]. The parameters were set as follows: zero or one occurrence per sequence, a motif width of 2–300, and a maximum number of 15 identified motifs. The other parameters had the default settings. The 2000 bp genomic sequences upstream of the transcription start site (ATG) of each SlRBZ-coding sequence were obtained from the tomato database. The PlantCARE online database was employed to analyze the putative cis-regulatory elements in the promoter regions of the SlRBZ genes [26].

2.5. Phylogenetic Tree and Duplication Event of SlRBZ Genes

The information regarding the RBZ proteins in Arabidopsis was obtained from Gipson et al. (2020) [10]. The RBZ protein sequences obtained from Arabidopsis and tomato for the phylogenetic analysis were separately obtained from TAIR (https://www.arabidopsis.org/, accessed on 20 October 2022) and the SGN database. The phylogenetic tree was constructed to show the phylogenetic relationships between the RBZ genes by the neighbor-joining method and maximum-likelihood method using the MEGA 7 software. A downloaded version of the MapChart tool was used to obtain the chromosomal location of the SlRBZ genes. To calculate the duplication event of the SlRBZ genes, the nonsynonymous substitution rates (Ka) and the synonymous rates (Ks) were calculated using KaKs_Calculator [27,28]. Calculated as T = Ks/2λ, the clock-like rate (λ) for the tomato was 1.5 × 10−8 substitutions/synonymous site/year [27].

2.6. RNA Sequencing Data Analysis

The data obtained from the transcriptomic analysis of various tissues in the tomato cultivar variety Heinz 1706 and the wild variety Solanum pimpinellifolium LA1589 were downloaded from the TFGD website (http://ted.bti.cornell.edu, accessed on 20 October 2022). The transcriptome data (raw data) of tomatoes under different abiotic stresses were also downloaded from the NCBI database, including cold (PRJEB14805), heat (PRJNA635375), drought (PRJNA635375) and salt (PRJNA624032) stress, and then reanalyzed. Clean data were obtained by removing the reads containing an adapter or ploy-N, as well as those of a low quality, and then were aligned with the reference genome (https://solgenomics.net/ftp/genomes/Solanum_lycopersicum/annotation/ITAG 2.4_release/, accessed on 20 October 2022) using Hisat2. FeatureCounts was used to count the read numbers mapped to each gene. Subsequently, TBtools was employed to calculate the fragments per kilobase of the exon model per million mapped fragments (FPKM) of each gene and create a heatmap [29].

3. Results

3.1. Identification and Isolation of the SlRBZ Genes in Tomato

Two methods were used to identify the members of the SlRBZ gene family in tomato. The first was to perform a BLAST search against the tomato genome using the SlRBZ6 sequences as queries. The second was to employ the consensus RanBP2 Znf domain sequences in the HMMER 3.0 program. Finally, 22 predicted SlRBZ genes were identified in the tomato genome. A detailed physicochemical analysis of the putative SlRBZ proteins revealed that the amino acid length and predicted MW ranged widely from 142 (aa)/15.45 kDa (SlRBZ19) to 1011 (aa)/111.53 kDa (SlRBZ15). The PI varied from 5.21 (SlRBZ16) to 10.87 (SlRBZ11). SlRBZ19 was the minimum and SlRBZ15 was the maximum member of the subfamily, respectively. It was found that two SlRBZ proteins, SlRBZ10 and SlRBZ12, were hydrophobic due to their positive GRAVY values, while the others were hydrophilic. The number of RanBP2 Znf domains ranged from one to four. More detailed information about the 22 SlRBZ genes is provided in Table 1. Coding sequences of the SlRBZ genes from the tomato Ailsa Craig were amplified and sequenced. The results are presented in File S2. All but four SlRBZ genes (SlRBZ2/10/15/19) matched the reference sequences. The introns of the SlRBZ2 and SlRBZ19 were not cleaved. The CDS length of SlRBZ10 was 146 bp shorter than the reference sequences. Interestingly, SlRBZ15 contained four isoforms in Ailsa Craig (Figure S1). Examining the number and sequence differences of the RanBP2 Znf domains can provide insight into their functions in tomato. A single RanBP2 Znf domain sequence was extracted from the UniProt sequence to construct an evolutionary tree using MEGA 7 (Figure S2). We observed that the Znfs within the clade I SlRBZ genes in the furthest C-terminal position were grouped together (Figure S2).

3.2. Multiple Sequence Alignments and Gene Structure of SlRBZ Genes

Multiple sequence alignments were performed to examine the features of the 22 SlRBZ genes. The results indicated that the sequence similarity among the SlRBZ genes ranged widely from 30.27% to 85.46% at the nucleotide level and from 0% to 87.23% at the amino acid level (Table S2). A neighbor-joining phylogenetic tree was constructed to investigate the evolutionary relationships between the SlRBZ members (Figure 1A). The 22 SlRBZ genes were divided into three clades: I, II and III. The formation of paralogous gene pairs (SlRBZ10/SlRBZ12 and SlRBZ1/SlRBZ16) had a strong bootstrap support (100%). The homology at the nucleotide level and amino acid level was 72.08% and 43.36%, and 60.06% and 49%, respectively (Table S2). The GSDS online server was employed to analyze the intron/exon structures of the SlRBZ genes. The exon number ranged from 2 to 17 (Table 1). Of the 22 SlRBZ genes, SlRBZ6/7/17 had 7 exons; SlRBZ3/4 had 5 exons; SlRBZ5/21 had 4 exons; SlRBZ10/11/14/18/20 had 3 exons; SlRBZ1/2/12/19 had 2 exons; and SlRBZ8/9/13/15/16/22 had 17, 10, 6, 16, 15 and 8 exons, respectively.

3.3. Conserved Motif Identification of SlRBZ Proteins

The MEME web server was used to predict the conserved motifs of the 22 deduced SlRBZ protein sequences. In total, we identified 15 distinct motifs (Figure 1B,D). The closest members in the phylogenetic tree, including two homologous gene pairs, always shared a common motif composition, which suggested an evolutionarily conserved and functional similarity within the same family. Meanwhile, some special motifs with unknown functions were identified. Motifs 1, 2, 9 and 4 fully matched the typical RanBP2 Znf domain of the SlRBZ genes based on our analyses of the Pfam and SMART databases. All these SlRBZ proteins contained Motifs 1 or Motif 2, or a combination of Motifs 9 and 4. Other motifs exhibited uniqueness in terms of some proteins, such as Motif 6 in SlRBZ4, SlRBZ7 and SlRBZ13; Motifs 7 and 3 in SlRBZ10 and SlRBZ12; and Motifs 5, 10, 11, 13 and 15 in SlRBZ1 and SlRBZ16. The differing compositions of the conserved motifs may be responsible for their functional diversity.

3.4. Evolutionary Relationships and Gene Duplication of the SlRBZ Family Members

To investigate the evolutionary relationships between RBZ genes from different plant species, a maximum-likelihood phylogenetic tree was constructed based on the alignment of 46 RBZ amino acid sequences from Arabidopsis and tomato (Figure 2). Most of the SlRBZ genes, including two paralogous gene pairs (SlRBZ10/SlRBZ12 and SlRBZ1/SlRBZ16), were separated by the Arabidopsis RBZ genes. This indicates that the genes originated before the split between tomato and Arabidopsis. The SlRBZ18/19/20 genes were clustered around each other and were not separated by Arabidopsis genes. A similar situation occurred in four of the ARI genes in Arabidopsis. The result indicated that the genes possibly emerged after the radiation of Arabidopsis and tomato.
The SlRBZ genes were unevenly distributed on 8 of the 12 tomato chromosomes (Figure 3). The number of SlRBZ genes appearing on each chromosome had a wide range. There were five SlRBZ genes on Chromosome 12 and four SlRBZ genes on Chromosome 1. Three SlRBZ genes were located on Chromosomes 3, 5 and 8, respectively, while there were two on Chromosome 11 and one on Chromosomes 2 and 7, respectively. Two gene pairs (SlRBZ1/16 and SlRBZ10/12) dispersed in the genome were identified as being involved in segmental duplication events. This trend suggests that segmental duplication, rather than tandem duplication, contributes to the expansion of the SlRBZ genes in tomato.
To assess the kinds of selection pressures among the duplicated SlRBZ genes, KaKs_Calculator was used to calculate the nonsynonymous (Ka) and synonymous substitutions (Ks) of the SlRBZ gene pairs (Table 2). The higher the Ks value is, the later the duplication events occurred. In this study, the Ka/Ks value for SlRBZ1/16 was 0.17 (<1), indicating that the gene pairs developed through purifying selection in the tomato. However, the Ka/Ks value for SlRBZ10/12 was 3.00 (>1), meaning that the gene pair developed through positive selection. The analysis of the dates of the duplication blocks showed that the duplication events of the SlRBZ genes were dated between approx. 31.60 and 69.83 million years ago (Mya), indicating that the SlRBZ genes expanded after the tomato/Arabidopsis division (~90 Mya).

3.5. Cis-Regulatory Elements in the Promoter Regions of SlRBZ Genes

It is apparent that cis-elements play an important role in transcription control. The level of gene expression is largely dependent upon the regulation of cis-elements in different organisms. Therefore, cis-elements must be analyzed in order to predict their biological functions. Genomic sequences found 2 kb upstream of the 5′ UTR of SlRBZ genes were queried in the PlantCARE database to search for putative cis-elements. The results showed that 94 putative cis-elements were present in the promoters (Table S3). Among these, two cis-elements, CAAT-box and TATA-box, were identified in all the SlRBZ genes. Overall, 18 cis-elements could be found in only one gene, including the AAAC-motif in SlRBZ22, GC-motif in SlRBZ11, MSA-like motif in SlRBZ11, AC-I motif in SlRBZ10 and AT-rich element in SlRBZ18, possibly endowing them with different biological functions. The result showed that these cis-elements can be divided into four types: stress responsiveness, light responsiveness, hormone responsiveness and plant growth and development (Figure 4).
The light responsiveness group, including Box 4, the TCT-motif, G-Box and GT1-motif, were found in most of the SlRBZ genes. LS7, the ATCT-motif and the AAAC-motif were located in SlRBZ9, SlRBZ11 and SlRBZ22, respectively. Additionally, we found 13 hormone responsiveness elements: ABRE (abscisic-acid (ABA)-responsive element); AT~ABRE and ERE (ethylene (ETH)-responsive elements); the CGTCA-motif and TGACG-motif (jasmonic-acid (JA)-responsive elements); TCA-element (salicylic-acid (SA)-responsive elements); GARE-motif, P-box and TATC-box (gibberellin (GA)-responsive elements); AuxRR-core and TGA-element (indole-3-acetic acid (IAA) responsiveness); and MYC and Myc, as displayed in the Supplementary Materials, Table S3. These results suggest that the transcription levels of the SlRBZ genes may be induced by these hormones. Furthermore, nine stress responsiveness elements were found, including MBS (drought inducibility), TC-rich repeats, MBSI and STRE (stress), ARE and GC-motif (anaerobic induction), LTR (low temperature), and WUN-motifs and WRE3 (wound responsiveness). In the plant growth and development group, some cis-elements are responsible for tissue-specific expressions, such as CAT-box in meristem cells, RY-element in the seeds, HD-Zip 1 in palisade mesophyll cells and GCN4 motif in the endosperms. These results indicate that SlRBZ genes might be involved in the complex regulatory networks during plant growth and development.

3.6. Tissue-Specific Expression Patterns of SlRBZ Genes

The organ/tissue-specific expression patterns of the 22 SlRBZ genes were investigated using bioinformatic and experimental approaches. The results are shown in a heatmap based on a publicly available RNA-seq library (Figure 5). All the SlRBZ genes, except for SlRBZ1/14/18/19/20, were constitutively expressed in all the examined tissues of Heinz (1706) (Figure 5A). Several genes in LA1589, such as SlRBZ1/13/14/18/19/20, were not expressed in certain tissues. The transcription levels of SlRBZ18 were abundant in the vegetative tissues, including the hypocotyls, cotyledons, vegetative meristems and mature leaves, and were barely expressed in the reproductive tissues (Figure 5B). The SlRBZ6/9/11/16 genes were consistently and highly expressed in Heinz (1706) and LA1589.
The qRT-PCR was performed to analyze the tissue expression patterns of 13 tomato SlRBZ genes that only have the RanBP2 Znf domain, without other known domains. All of the 13 SlRBZ genes were constitutively expressed in all the examined tissues (Figure 6). All the SlRBZ genes in tomato, except for SlRBZ10, had the highest relative abundance in the flowers or leaves. Nevertheless, the transcription levels of SlRBZ10 were more abundant in the green mature stage. SlRBZ3/9/13/18/19/20 showed similar expression patterns. The expression of the genes was highest in the leaves, followed by the red, ripe fruits, it was lowest in the immature fruits. During fruit development and ripening, the transcription levels of most of SlRBZ genes steadily increased; therefore, these genes may function in the late-stage development of tomatoes. In contrast, SlRBZ10/11/14 decreased during fruit development, indicating that these three genes are involved in the early stage of the development of tomato fruits. Importantly, SlRBZ11 was the only gene whose expression was lowest in the leaves.

3.7. Expression Pattern of SlRBZ Genes under Different Phytohormones

Phytohormones play a crucial role in controlling the plant growth, development and response to environmental stimuli. Previous studies showed that the expression of RBZ genes can be induced by ABA and GA [6,14,15]. To determine whether the tomato SlRBZ genes can be induced by hormones, we conducted a qRT-PCR using the materials obtained after treatments with several plant hormones. SlRBZ genes have various responses to different plant hormones (Figure 7). SlRBZ3/9/14/19 genes were highly induced by the ethephon treatment. The SlRBZ9/14 expression peaked at ETH-3 h, while the SlRBZ3/19 expression peaked at ETH-9 h. The transcription levels of these genes were 41.65 to 3061.45 times higher than those of the mock treatment. The SlRBZ7/13 genes were induced at GA-9 h by 4.86 and 116.70 times. Some genes were induced by JA, such as SlRBZ4/10, which was induced by JA-9 h by 2463.80 and 148.40 times. SlRBZ6 was also induced slightly by JA-24 h by 3.60 times. SlRBZ2/20 was induced strongly by 6BA-12 h by 10,297.45 and 186,222.30 times. The SlRBZ18 expression peaked at ABA-12 h after treatment by about 166.96 times. The SlRBZ11 expression peaked at SA-12 h by approximately 6.89 times. SlRBZ6 was not sensitive to these hormones (Figure S3).

3.8. Expression Characteristics of the SlRBZ Genes under Various Abiotic Stresses

To assess the potential functions of the SlRBZs in response to different abiotic stresses (cold, heat, drought and salt), a transcriptome analysis, combined with a qRT-PCR assay, was employed (Figure 8 and Figure 9). Under the chilling treatment, the expression of SlRBZ6/9/13/15/16 genes exhibited a decreasing trend in response to the 15 ℃ treatment in the case of LA1777 and Moneymaker. In contrast, the other genes were insensitive to the chilling treatment. The SlRBZ genes exhibited three distinct patterns under heat stress. Whereas SlRBZ3/4/7/9/17/18 showed an obvious upward trend, SlRBZ14/15 showed an obvious downward trend. SlRBZ16 showed a downward trend first and then an upward trend. About 50% of the SlRBZ genes (11 of 22) were induced by drought stress. Seven SlRBZ genes (SlRBZ6/8/10/14/16/18/22) were significantly suppressed by salt. However, the SlRBZ2/3/4/13 genes were induced by salt at 8 h.
The transcript profiles of 13 SlRBZ genes, which only have the RanBP2 Znf domain, without other known domains, were analyzed under different abiotic stresses by qRT-PCR analysis (Figure 9). All the examined SlRBZ genes could be induced differently by more than one abiotic stress. Most of the examined SlRBZ genes were strongly induced by salt, with the highest transcription levels observed at 0.5 h after treatment. Among the genes, the SlRBZ20 expression peaked at salt-0.5 h, with an expression 2.43e4 times higher than that of the mock treatment. SlRBZ6/15 were induced by salt at 12 h and 24 h, respectively. All the examined SlRBZ genes, except for SlRBZ6/13/18/19, were positively affected by cold, heat and drought. The SlRBZ6 transcription levels remained unchanged under cold and drought stress conditions. Interestingly, SlRBZ13/18/19 were downregulated in response to cold and heat treatments, as well as drought treatment. The expression levels of SlRBZ9 were increased by all four treatments, with the highest induction observed in response to the drought and salt treatments (approximately 12 times). The transcription level of SlRBZ11 was highly increased at 6 h after heat stress, with an expression 18.68 times that of the mock treatment.

4. Discussion

RBZ transcription factors are common in animals and plants. This type of transcription factor was first discovered in the nuclear export protein RanBP2 and is involved in mRNA processing in humans [30,31]. However, it has not yet been widely characterized in plants. Similar to other zinc finger proteins, RBZ transcription factors are highly conserved between species. The number of RBZ family members in tomato is similar to that in Arabidopsis. It has been reported that the expansion of gene families results from gene duplication events, including segmental and tandem duplications [32]. The duplicated genes start with the same sequence and then develop different regulatory and coding regions [33]. Segmental duplication likely contributed to the expansion of the SlRBZ gene family.
The multiple sequence alignment results show that the sequence similarity of the SlRBZ genes at the nucleotide level was 30.27~85.46% and the similarity at the amino acid level was 0~87.23% based on the multiple sequence alignments. Therefore, the SlRBZ genes were highly complex, according to the widely varied nucleotide sequences’ lengths and exon numbers. This suggests that SlRBZ genes may be involved in different biological processes. Accordingly, the SlRBZ proteins contain various conserved motifs based on the motif analysis using the MEME network. The examination of the number and sequence differences between the RanBP2 Znf domains can provide insight into their functions within the full-length protein. In this study, the number and sequence differences between the RanBP2 Znf domains on the SlRBZ genes were also examined. Our results show that the number of RanBP2 Znf domains ranges from 1 to 4, and the sequence is well conserved across its different iterations. This supports earlier studies involving Arabidopsis [10]. Alternative splicing is an important transcriptional regulatory mechanism and is widely found in eukaryotes. Previous studies have shown that 95% of the human genes containing multiple exons have alternative splicing [34]. There are many splicing types, including the skipped exon types, intron retention, competing 5′ splice sites, etc. Our results show that SlRBZ2, SlRBZ15 and SlRBZ19 may undergo intron retention. Similarly, alternative splicing events of the RBZ genes were also found in Arabidopsis [10]
The characterization of genes’ expression patterns reveals their regulatory roles in plant growth and development. Our results show that all the examined SlRBZ genes were constitutively expressed in the eight examined tissues. HDA15 is highly expressed in the stems throughout the life of Arabidopsis [35]. AtRBL10 is expressed consistently across the plant tissues and their development, and it plays a positive role in heat shock [14]. The transcription levels of SlRBZ10 (the orthologous gene of AtRBL10) were more abundant in the green mature stage and became progressively lower during fruit development, thus indicating that SlRBZ10 plays a role in the early stage of the development of tomato fruits. Furthermore, the SlRBZ10 expression was significantly upregulated following heat treatment in this study. This indicated that SlRBZ10 and AtRBZ10 may have similar functions in response to heat shock. In this study, the transcription levels of SlRBZ6 were induced by the eight examined phytohormones by 4-fold less than the control induced by qRT-PCR. Fan et al. (2015) reported that SlRBZ6 was induced by GA-24 h and IAA-1 h by five-fold less than the control and was not sensitive to ABA [6]. This is similar to our experimental results.
Plant hormones play important roles in various abiotic and biotic stresses during plant development [36]. Abiotic stress (including extreme temperature, high salinity and drought) is the leading cause of crop loss worldwide, resulting in average yield declines of more than 50% in most major crops. In this study, most of the SlRBZ genes were induced by hormones or abiotic stress, which was consistent with the prediction regarding their promoter cis-regulatory elements. For example, the expression level of SlRBZ2/14/18/19/20 was high under the ABA treatment (Figure 7). SlRBZ3/18/20 was induced to varying degrees by GA. SlRBZ3/10 was induced 9 h after the cold treatment, confirming the predicted low-temperature responsiveness of the cis-regulatory elements in the promoter region (Table S3). This indicated that SlRBZ3/10 is likely to be involved in the cold–stress response. The expression level of SlRBZ2/7/9/10/13/18/19 was induced after the drought treatment, supporting the predicted drought responsiveness of the cis-regulatory elements in the promoter region (Table S3).
Both a transcriptome analysis and qRT-PCR assay were employed to assess the potential functions of the SlRBZs in response to different abiotic stresses. The transcription levels of SlRBZ13 were suppressed by cold, SlRBZ19 was suppressed by drought and SlRBZ9 was induced by heat and drought in the qRT-PCR analysis (Figure 9). These results were consistent with the changes in the RNA-seq data. SlRBZ6/20 were induced by salt at 12 h and 0.5 h in the qRT-PCR analysis (Figure 9). However, SlRBZ6 was suppressed by salt based on the RNA-seq data. There was no obvious change in the expression of SlRBZ20 after the salt treatment based on the RNA-seq data (Figure 8). Therefore, the results do not confirm the changes in the RNA-seq data. This difference might be caused by the different materials or different stress treatment times and methods employed.

5. Conclusions

This study undertook a comprehensive, genome-wide gene identification of the RBZ family in tomato. A total of 22 SlRBZ genes were identified. We analyzed the phylogeny, conserved motifs, gene structure, chromosomal location, cis-elements and expression patterns of all the SlRBZ genes in different tissues from tomato using bioinformatics methods. A qPCR analysis was performed to analyze the tissue expression levels of the SlRBZ genes in tomato to induce the expression levels under the effects of eight different hormones and four abiotic stress treatments. The results showed that most of the SlRBZ genes responded to at least one abiotic stress or plant hormone, thereby indicating their potential functions in such processes. In general, this research provides comprehensive information and a basis for future functional research on the SlRBZ genes in tomato.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae8110985/s1, File S1. The nucleotide and amino acid sequences of 22 identified SlRBZ genes downloaded from the SOL Genomics Network. File S2. The coding sequences of 22 identified SlRBZ genes in Ailsa Craig. Figure S1. Gene structure of different transcripts of SlRBZ15. Figure S2. Phylogenetic tree of tomato RanBP2 zinc finger domains. Figure S3. Expression profiles of SlRBZ6 under exogenous phytohormone treatments. Table S1. Primers used in this study. Table S2. Multiple Sequence Alignments were carried out with the ClustalW2 web server using the nucleotide (nt)a and amino acid (aa)b sequences of the SlRBZ genes. Table S3. A total of 94 cis-elements identified using PlantCARE program in the promoter regions of the SlRBZ genes.

Author Contributions

Y.G. designed the experiments, carried out the inducing of the expression profiles, analyzed the data and drafted the manuscript. J.R. and N.L. carried out the amplification of the sequences of SlRBZ genes and analyzed the data. N.L. analyzed the cis-regulatory elements within the promoters and conserved motifs of the SlRBZ genes. X.L. and Y.L. performed the qRT-PCR for the tissue-preferential expression profiles of the SlRBZ genes and analyzed the data. C.Y. designed the experiments and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 31801880). The Key Research and Development Foundation of Henan (No. 222102110407).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic tree (A), conserved motif distribution (B) and sequences (D), and gene structure (C) of the SlRBZ genes. Motif 1 or Motif 2, or a combination of Motifs 9 and 4, represent the typical RanBP2 Znf domain.
Figure 1. Phylogenetic tree (A), conserved motif distribution (B) and sequences (D), and gene structure (C) of the SlRBZ genes. Motif 1 or Motif 2, or a combination of Motifs 9 and 4, represent the typical RanBP2 Znf domain.
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Figure 2. Phylogenetic tree of 46 RBZ genes from tomato and Arabidopsis.
Figure 2. Phylogenetic tree of 46 RBZ genes from tomato and Arabidopsis.
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Figure 3. Chromosomal location of SlRBZ genes. The gene pairs are linked by dashed lines.
Figure 3. Chromosomal location of SlRBZ genes. The gene pairs are linked by dashed lines.
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Figure 4. Information on cis-acting elements in the putative promoter regions of SlRBZs. (A) The gradient colors in the red grid indicate the numbers of cis-acting elements in the putative promoter regions of SlRBZs. (B) The X-axis shows the number of different types of cis-elements.
Figure 4. Information on cis-acting elements in the putative promoter regions of SlRBZs. (A) The gradient colors in the red grid indicate the numbers of cis-acting elements in the putative promoter regions of SlRBZs. (B) The X-axis shows the number of different types of cis-elements.
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Figure 5. Heatmap of the expression profiles of SlRBZ genes in the cultivated tomato cultivar Heniz 1706 (A) and the wild species S. pimpinellifolium LA1589. (B) Fully opened flowers (FOF), unopened flower buds (UFB), 1 cm fruits (1cm.F), 2 cm fruits (2cm.F), 3 cm fruits (3cm.F), mature green fruits (MGF), breaker fruits (BR), breaker+10 fruits (BR+10F), roots (RT), leaves (L), days post-anthesis fruit (DPA), cotyledons (COTYL), hypocotyl (HYPO), vegetative meristems (MERI), mature leaves (ML), young flower buds (YFB), young leaves (YL). Dark red indicates higher expression levels, and dark blue indicates lower expression levels. Genes with similar expression profiles across various arrays are grouped on the left based on a hierarchical clustering method.
Figure 5. Heatmap of the expression profiles of SlRBZ genes in the cultivated tomato cultivar Heniz 1706 (A) and the wild species S. pimpinellifolium LA1589. (B) Fully opened flowers (FOF), unopened flower buds (UFB), 1 cm fruits (1cm.F), 2 cm fruits (2cm.F), 3 cm fruits (3cm.F), mature green fruits (MGF), breaker fruits (BR), breaker+10 fruits (BR+10F), roots (RT), leaves (L), days post-anthesis fruit (DPA), cotyledons (COTYL), hypocotyl (HYPO), vegetative meristems (MERI), mature leaves (ML), young flower buds (YFB), young leaves (YL). Dark red indicates higher expression levels, and dark blue indicates lower expression levels. Genes with similar expression profiles across various arrays are grouped on the left based on a hierarchical clustering method.
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Figure 6. Expression profiles of SlRBZ genes in different tissues and organs. RT, root; ST, stem; YL, young leaf; FL, flower; IM, pericarp of immature fruit; GM, pericarp of mature green fruit; BR, pericarp of breaker fruit; RR, pericarp of red ripe fruit. Error bars represent standard deviations for the three replicates.
Figure 6. Expression profiles of SlRBZ genes in different tissues and organs. RT, root; ST, stem; YL, young leaf; FL, flower; IM, pericarp of immature fruit; GM, pericarp of mature green fruit; BR, pericarp of breaker fruit; RR, pericarp of red ripe fruit. Error bars represent standard deviations for the three replicates.
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Figure 7. Expression profiles of the SlRBZ genes under exogenous phytohormone treatments. The numbers (1–9) below the x-axis represent the time course of the hormone treatment, e.g., for 1: 0.25 h; 2, 0.5 h; 3, 1 h; 4, 3 h; 5, 6 h; 6, 9 h; 7, 12 h; 8, 24 h; 9, 36 h. Here, the number is the interval, displayed for clarity. The y-axis represents the relative transcription levels of the SlRBZ genes compared to the mock, which was sprayed with water. Error bars represent standard deviations for the three replicates.
Figure 7. Expression profiles of the SlRBZ genes under exogenous phytohormone treatments. The numbers (1–9) below the x-axis represent the time course of the hormone treatment, e.g., for 1: 0.25 h; 2, 0.5 h; 3, 1 h; 4, 3 h; 5, 6 h; 6, 9 h; 7, 12 h; 8, 24 h; 9, 36 h. Here, the number is the interval, displayed for clarity. The y-axis represents the relative transcription levels of the SlRBZ genes compared to the mock, which was sprayed with water. Error bars represent standard deviations for the three replicates.
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Figure 8. Expression patterns of SlRBZ genes under abiotic stresses based on RNA-seq. Moneymaker (M) and LA1777 (LA) were treated for 3 d at 23 ℃ and 15 ℃. Under heat and drought conditions, the seedlings of Solanum lycopersicum cv M82 were kept under 42 ℃ and the drought treatment, respectively. In salt, the seedlings of Micro-Tom were sprayed with 200 mM NaCl.
Figure 8. Expression patterns of SlRBZ genes under abiotic stresses based on RNA-seq. Moneymaker (M) and LA1777 (LA) were treated for 3 d at 23 ℃ and 15 ℃. Under heat and drought conditions, the seedlings of Solanum lycopersicum cv M82 were kept under 42 ℃ and the drought treatment, respectively. In salt, the seedlings of Micro-Tom were sprayed with 200 mM NaCl.
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Figure 9. Expression patterns of SlRBZ genes under abiotic stresses by qRT-PCR. Error bars represent standard deviations for the three replicates.
Figure 9. Expression patterns of SlRBZ genes under abiotic stresses by qRT-PCR. Error bars represent standard deviations for the three replicates.
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Table
Table 1. Description of SlRBZ genes in tomato.
Table 1. Description of SlRBZ genes in tomato.
GeneAnnotated CDSGene Start (bp)Gene End (bp)Strandthe Number of RBZ DomainsExonORF * CDS *AA *PIS *MW *GRAVYDomains
SlRBZ1Solyc01g005650455379456988−112161015695235.3661478.64−0.709RBZ, IBR domain
SlRBZ2Solyc01g04435043255687432593711229698942989.1231513.46−0.432RBZ
SlRBZ3Solyc01g0577806386286263868831−125556426258756.9997971.39−0.723RBZ
SlRBZ4Solyc01g0992308953033489535887125523323527846.6689729.05−1.11RBZ, MSCRAMM_SdrC super family
SlRBZ5Solyc02g0328702943897029441887124270714194737.9752898.98−0.351RBZ, TDP2
SlRBZ6Solyc03g03356051043845111275−14765109963329.0136450.1−0.63RBZ
SlRBZ7Solyc03g1186806753826467542652−137399412244088.9446033.82−0.555RBZ
SlRBZ8Solyc03g11973068283528682920671117832618336115.3466410.65−0.274RBZ, HDAC_classII
SlRBZ9Solyc05g0155001076027110764983131043508582869.1430923.51−0.859RBZ
SlRBZ10Solyc05g0163801603473116038070113307811163728.4642409.010.156RBZ
SlRBZ11Solyc05g01834020573843205799111135800105635210.8739607.39−1.519RBZ
SlRBZ12Solyc07g042930564857575648839111221259963329.8237398.510.057RBZ, Rhomboid
SlRBZ13Solyc08g01451046452154647952−136245214434818.4655095.32−0.756RBZ
SlRBZ14Solyc08g0671805615534656156822−1338785131718.7118130.78−0.782RBZ
SlRBZ15Solyc08g0773106120940361219976111610300303310117.96111526.92−0.983RBZ, RRM, OCRE domain, G_patch
SlRBZ16Solyc11g00858027648232775320−11151049817765925.2166955.47−0.659RBZ, Ring finger
SlRBZ17Solyc11g0400504002444740028245127379911703908.6243070.15−1.155RBZ, RRM_SARFH
SlRBZ18Solyc12g006590107964510802871336434381468.20 16061.15−0.537RBZ
SlRBZ19Solyc12g006600108378610843091325244261428.415446.45−0.472RBZ
SlRBZ20Solyc12g0110603931559393270913311514981668.6118535.23−0.41RBZ
SlRBZ21Solyc12g01566056558555659081−124322712454158.745811.89−0.626RBZ, WLM domain
SlRBZ22Solyc12g0364105219355152201012−128746218036018.5965495.48−0.168RBZ, translation elongation factor EF-1a (GTPase)
* ORF, length of open reading frame (number of nucleotides); CDS, length of CDS; AA, protein length (number of amino acid); PIS, theoretical isoelectric point; MW, molecular weight, KDa.
Table
Table 2. Duplicated genes and the dates of the duplication blocks of SlRBZ genes.
Table 2. Duplicated genes and the dates of the duplication blocks of SlRBZ genes.
Gene1Gene2KsKaKa/KsDate (Mya)
SlRBZ1SlRBZ160.952.84331.6
SlRBZ10SlRBZ122.090.360.1769.83
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Gao, Y.; Li, N.; Ruan, J.; Li, Y.; Liao, X.; Yang, C. Genome-Wide Identification, Cloning and Expression Profile of RanBP2-Type Zinc Finger Protein Genes in Tomato. Horticulturae 2022, 8, 985. https://doi.org/10.3390/horticulturae8110985

AMA Style

Gao Y, Li N, Ruan J, Li Y, Liao X, Yang C. Genome-Wide Identification, Cloning and Expression Profile of RanBP2-Type Zinc Finger Protein Genes in Tomato. Horticulturae. 2022; 8(11):985. https://doi.org/10.3390/horticulturae8110985

Chicago/Turabian Style

Gao, Yanna, Ning Li, Jiaojiao Ruan, Ying Li, Xiaoli Liao, and Changxian Yang. 2022. "Genome-Wide Identification, Cloning and Expression Profile of RanBP2-Type Zinc Finger Protein Genes in Tomato" Horticulturae 8, no. 11: 985. https://doi.org/10.3390/horticulturae8110985

APA Style

Gao, Y., Li, N., Ruan, J., Li, Y., Liao, X., & Yang, C. (2022). Genome-Wide Identification, Cloning and Expression Profile of RanBP2-Type Zinc Finger Protein Genes in Tomato. Horticulturae, 8(11), 985. https://doi.org/10.3390/horticulturae8110985

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