- Research article
- Open Access
Identification of alternative splicing events by RNA sequencing in early growth tomato fruits
© Sun and Xiao. 2015
- Received: 13 July 2015
- Accepted: 22 October 2015
- Published: 16 November 2015
Alternative splicing (AS) regulates multiple biological processes including flowering, circadian and stress response in plant. Although accumulating evidences indicate that AS is developmentally regulated, how AS responds to developmental cues is not well understood. Early fruit growth mainly characterized by active cell division and cell expansion contributes to the formation of fruit morphology and quality traits. Transcriptome profiling has revealed the coordinated complex regulation of gene expression in the process. High throughput RNA sequencing (RNA-seq) technology is advancing the genome-wide analysis of AS events in plant species, but the landscape of AS in early growth fruit is still not available for tomato (Solanum lycopersicum), a model plant for fleshy fruit development study.
Using RNA-seq, we surveyed the AS patterns in tomato seedlings, flowers and young developing fruits and found that 59.3 % of expressed multi-exon genes underwent AS in these tissues. The predominant type of AS events is intron retention, followed by alternative splice donor and acceptor, whereas exon skipping has the lowest frequency. Although the frequencies of AS events are similar among seedlings, flowers and early growth fruits, the fruits generated more splice variants per gene. Further comparison of gene expression in early growth fruits at 2, 5 and 10 days post anthesis revealed that 5206 multi-exon genes had at least one splice variants differentially expressed during early fruit development, whereas only 1059 out of them showed differential expression at gene level. We also identified 27 multi-exon genes showing differential splicing during early fruit growth. In addition, the study discovered 2507 new transcription regions (NTRs) unlinked to the annotated chromosomal regions, from where 956 putative protein coding transcripts and 1690 putative long non-coding RNAs were identified.
Our genome-wide analysis of AS events reveals a distinctive AS pattern in early growth tomato fruits. The landscape of AS obtained in this study will facilitate future investigation on transcriptome complexity and AS regulation during early fruit growth in tomato. The newly found NTRs will also be useful for updating the tomato genome annotation.
- Alternative splicing
- New transcription regions (NTRs)
Tomato (S.lycopersicum) is a model plant for studying fleshy fruit development. Fruit development post anthesis can be divided into four major stages—fruit set, cell division, cell expansion and ripening [1–3]. Early fruit growth in tomato, usually referring to the growth within 2–3 weeks after anthesis, is characterized by rapid cell division and drastic cell expansion. For example, in the small-fruited wild tomato species S.pimpinellifolium the cell number of pericarp is doubled within 2 days post anthesis (dpa) and cell division ceases around 5 dpa, whereas cell expansion starting as early as 2 dpa contributes to the remaining fruit growth . Therefore, fruit size largely determined by cell number and cell size is defined predominantly during early fruit growth. In addition, cell expansion during early fruit growth also contributes to the major changes in fruit structure and its biochemical and physiological properties. Several genes regulating the formation of important agricultural traits in tomato have been shown to exert their actions during early fruit growth . For example, ovate and sun, two major quantitative trait loci (QTLs) controlling fruit shape and the major fruit weight locus fw2.2 execute their functions mainly during early fruit development [5–7]. The cell wall invertase gene LIN5 also regulates the formation of solid soluble content during early fruit growth . Several studies of gene expression profiling using microarray have revealed that during tomato fruit set and early growth more than 1000 genes are differentially expressed [2, 9–12], indicating that complex transcriptional regulation, especially of genes related to cell division and hormone biosynthesis and perception, is involved in early fruit growth.
Regulation of gene expression is perceived at multiple levels including transcriptional and post-transcriptional regulation. Alternative splicing (AS) of precursor message RNA (pre-mRNA), which can generate a number of different transcripts from individual multi-exon genes, is one of the important regulatory mechanisms at post-transcriptional level in eukaryotes [13–16]. AS may increase transcriptome complexity and expand genome’s protein coding capacity, having a profound impact on protein functionality, stability and expression levels [15, 17]. By analysis of expressed sequence tags (EST) and RNA-seq data, AS events with various frequencies have been detected in many eukaryotes, including yeast, fungi, plants and animals [18–20]. For example, it has been estimated that more than 95 % of the human multi-exon genes undergo AS [21, 22]. In plants, it has been estimated that 40–63 % of multi-exon genes undergo AS [23–28]. Recently, Chamala et al. have performed a survey on AS patterns in different plant species by computational analysis of EST, mRNA and short reads from public available RNA-seq data, and found that depending on species, 39.1–70.4 % of multi-exon genes produce at least one splice variants in nine plant species including tomato (S.lycopersicum), Medicago truncatula, soybean (Glycine max), common bean (Phaseolus vulgaris), rice (Oryza sativa), Arabidopsis thaliana, poplar (Populus trichocarpa), grape (Vitis vinifera) and Amborella .
AS plays important roles in many biological processes, especially in response to environmental stresses . Also, AS is regulated by developmental processes and by external stimuli, such as light , temperature [32, 33], salt  and pathogens . Wang et al. using RNA-seq assessed the genome-wide changes of AS events during Arabidopsis flower development, and found that AS patterns are similar between different developmental stages . They reported that about 25 % of the expressed multi-exon genes underwent AS and 1716 splice variants were differentially expressed, indicating that AS is regulated by floral development. Developmental regulation of AS events has also been confirmed in maize leaf and poplar xylem [37, 38]. Despite the recent advance in identification of organ-specific AS events and in developmental regulation of AS in few model plants, the AS landscape during fruit development has not yet been obtained from tomato, a model plant for studying fleshy fruit development.
To investigate the AS patterns of early growth fruits, we applied strand-specific RNA-seq to analyze the genome-wide AS events in tomato tissues including seedlings, flowers and early growth fruits at 2, 5 and 10 dpa, and compared the AS patterns in fruits between three characteristic stages of early fruit growth. We found 59.3 % of expressed multi-exon genes underwent AS in at least one of these tissues investigated. Furthermore, the study revealed that there were more splice variants per gene detected in early growth fruits than in seedlings and flowers. We also identified 27 genes showing differential splicing during early fruit growth. In addition, we identified 2507 new transcription regions (NTRs) unlinked to any annotated chromosomal segments, providing a rich resource for future functional genomic analysis in tomato.
Identification of AS events in early fruit development
Summary of read mapping
Raw read pairs
Mapped read pairs
2 dpa fruit
5 dpa fruit
10 dpa fruit
To validate the AS events detected by RNA-seq, we performed a reverse transcription-PCR (RT-PCR) analysis on 22 multi-exon genes producing the five types of AS events using RNA samples isolated from seedlings, flowers and fruits at different developmental stages. We were able to detect the expression of these splice variants in multiple samples, and further found that several splice variants showed tissue-specific expression patterns (Fig. 4). For example, both of Solyc03g046470 and Solyc05g009360 had one splice variant only expressed in the fruits at 2, 5 and 10 dpa, but not in seedlings and flowers (Fig. 4). Moreover, the RT-PCR analysis showed that the AS events detected in the LA1589 samples were also found in seedlings or flowers of cultivated tomato, except those only expressed in fruit tissues.
Genes showing differential splicing during early fruit development
Gene showing differential splicing during early fruit development
Set A. Differential splicing between 2 and 5 DPA
26S proteasome regulatory subunit; Mov34/MPN/PAD-1
bisphosphoglycerate-dependent phosphoglycerate mutase; Phosphoglycerate mutase
RNA polymerase II C-terminal domain phosphatase-like 1; NLI interacting factor
T-complex protein 1 subunit gamma
Genomic DNA chromosome 5 P1 clone MWD9
Membrane-associated zinc metalloprotease family protein expressed; putative membrane-associated zinc metallopeptidase
Mitochondrial import inner membrane translocase subunit TIM14; Heat shock protein DnaJ, N-terminal
Ariadne-like ubiquitin ligase; Zinc finger, C6HC-type
DNA ligase; ATP-dependent DNA ligase
Set B. Differential splicing between 5 and 10 DPA
Mediator of RNA polymerase II transcription subunit 31; Mediator complex subunit Med31
Glucan endo-1 3-beta-glucosidase 5; Glycoside hydrolase subgroup, catalytic core
ZZ type zinc finger domain-containing protein (Fragment); Octicosapeptide/Phox/Bem1p
Calmodulin-binding protein MPCBP; Tetratricopeptide-like helical; similar to NO POLLEN GERMINATION RELATED 2, NPGR2 of Arabidopsis
Tetratricopeptide repeat protein 28; Tetratricopeptide-like helical
Set C. Differential splicing between 2 and 10 DPA
Serine/threonine-protein kinase 36; SlMAPKKK1
MORC family CW-type zinc finger 3; ATP-binding region, ATPase-like; Histidine kinase, DNA gyrase B-, and HSP90-like ATPase family protein
Integrin-linked kinase-associated serine/threonine phosphatase 2C; Protein phosphatase 2C
similar to AtCYO1, SCO2 | protein disulfide isomerases
Chromodomain helicase DNA binding protein 5; SNF2-related
Acyl-CoA oxidase 6; Acyl-CoA oxidase
Upf3 regulator of nonsense transcripts-like protein B; Regulator of nonsense-mediated decay UPF3
Ubiquitin carboxyl-terminal hydrolase family protein expressed; ubiquitin carboxyl-terminal hydrolase 2
Differentially expressed splice variants during early fruit growth
At gene level, there were 1945 genes differentially expressed (adjusted p-value smaller than 0.05) during early fruit growth, of which 396 and 1737 genes showed differential expression between two consecutive stages of early fruit development, respectively (Additional file 2: Table S1). But as shown in Fig. 5 and Additional file 1: Figure S1, some splice variants showed different expression patterns from its annotated transcript. We then investigated the expression patterns of individual isoforms in 2, 5 and 10 dpa fruits. In these young developing fruits, a total of 29,395 splice variants were detected. Expression level below 1 FPKM is thought to be beyond the limit of protein detection [46–48]. By applying the very conservative cutoff of expression values at 1 FPKM, we found a total of 12,804 splice variants were expressed in these tissues. Among them, there were 6198 and 6564 splice variants from a total of 5206 annotated multi-exon genes showing expression changes by four folds or higher between two consecutive stages at 2, 5 and 10 dpa; these splice variants were considered to be differentially expressed during early fruit growth (Additional file 3: Table S2). When applying the same criteria to the annotated transcripts, only 1059 out of the 5206 genes were differentially expressed, indicating that gene expression during early fruit growth is regulated at multiple layers and expression of splice variants is highly modulated (Additional file 4: Table S3).
Several fruit size and shape genes have been shown to exert their functions during early fruit growth. For example, the SUN (Solyc10g079240) gene encoding an IQD protein family member defines fruit shape early during fruit development and is expressed at higher levels concomitantly . Three isoforms of SUN mRNA were detected in the young fruits and the longest splice variant was downregulated after 2 dpa, in contrast to the constant expression of the annotated SUN transcript. The shorter SUN splice variant was resulted from altered transcription start site at the third intron, encoding a smaller protein, but expressed at low level (FPKM <1.0).
We further conducted an enrichment analysis of GO ontology on the two data sets containing the differentially expressed transcripts at gene and isoform levels, respectively. The analysis revealed that three functional categories—transporter activity (GO:0005,215), catalytic activity (GO:0003824) and hydrolase activity (GO:0016787)—were overrepresented in both of the two data sets (Fig. 6c and d). However, GO term of oxygen binding (GO:0019825) was only enriched in the data set showing differential expression at gene level, whereas two categories of protein binding (GO:0005515) and nucleotide binding (GO:0000166) were overrepresented in that with differentially expressed splice variants. At gene level, genes involved in two biological processes of carbohydrate metabolic process (GO:0005975) and transport (GO:0006810) were overrepresented, but there were no enriched GO terms of biological processes for these genes with differentially expressed splice variants.
Novel transcription regions
In addition, three quarters of the 2650 transcript contigs have nucleotides longer than 500 bp, indicating many of them are likely protein coding sequences. Indeed, blast research against NCBI database revealed that there were 819 NTR transcripts sharing high similarity (e value <1.0e-10) with protein coding sequences from various plant species. In addition, 4 and 474 transcript contigs share high sequence similarity with microRNA precursors and putative long non-coding RNAs (lncRNAs) annotated in NCBI database, respectively (Additional file 5: Table S4). The remaining 1353 NTR transcript contigs share no significant similarity with protein encoding sequences, but 137 of them contain open reading frames longer than 100 amino acids. Therefore, they are likely new protein coding genes (Additional file 6: “Novel protein sequences”). Thus, in addition to the 1690 putative lncRNAs, there are likely 956 new protein coding genes in the tomato genome that have not been predicted.
Recently developed high throughput sequencing technology has allowed effective genome-wide detection of AS events in several plant species. In this study, we used strand-specific RNA-seq to conduct a genome-wide analysis of AS events in cultivated tomato and its direct ancestor S.pimpinellifolium and compared the AS patterns between three characteristic stages of early growth fruits. We found that nearly 60 % of tomato multi-exon genes were alternatively spliced, a much higher frequency than the recently estimated 39.1 % . But the frequency is close to the highest ones discovered up to now in Arabidopsis and soybean [24, 25]. More AS events discovered in our analysis is likely benefited from the longer reads we used because longer reads apparently retain more sequence information of the splice junctions. It is also known that the AS frequencies discovered within the same plant species vary considerably among different experiments because of different tissue types and growth conditions as well as different prediction algorithms used. For example, in Arabidopsis, the frequencies of multi-exon genes that underwent AS range from 25 to 61 % [25, 36]. Since AS is affected by tissue types, developmental stages and growth conditions, we would expect higher AS frequencies to be detected in tomato when different tissues at various developmental stages and under different growth conditions are analyzed.
In tomato, early fruit growth contributes to the formation of fruit traits such as fruit morphology and sugar contents [2, 5, 8]. Fruit growth beginning with successful fertilization of ovules has a short duration of cell division followed by a long concomitant cell expansion phase . Transcriptomic profiling has identified a number of differentially expressed genes during fruit set and early fruit growth [2, 9–12]. The identification of 1945 genes differentially expressed in S.pimpinellifolium during very early fruit growth (from 2 to 10 dpa) further provides informative knowledge on gene regulation associated with early fruit growth, especially on cell division and cell expansion, which has not been targeted previously. Our gene profiling analysis also revealed that there were a much higher number of genes differentially expressed between 5 and 10 dpa. The result is in agreement with the previous report that from 2 to 5 dpa, S.pimpinellifolium fruits mainly undergo cell division, whereas developmental program is shifted to cell expansion in 10 dpa fruits . The expression patterns of genes involved in auxin, gibberellin and cytokinin pathways as well cell cycle regulation are also consistent with the cell division and differentiation in the fruits at the three time points.
Furthermore, our analysis discovered a distinctive AS pattern in early fruit growth, which more AS events per gene were identified in the young fruits compared to other tissues, although the frequencies of AS events in the fruits were close to those in seedlings and flowers. Compared to the number of differentially expressed genes at gene level, more genes showed differential expression at isoform level. We found that 5206 genes had at least one splice variants differentially expressed during early fruit growth, whereas only 1945 of them were differentially expressed at gene level. This indicates that AS plays an indispensible role in regulation of gene expression during early fruit growth. It is worthy to notice that although more genes were differentially expressed during the transition from cell division to cell expansion (5–10 dpa) at gene level, there were similar numbers of splice variants differentially expressed between cell division phase (2–5 dpa) and the cell division-expansion transition. We also identified 27 genes showing significant changes in AS patterns during early fruit growth. It has been shown that the abundance and activity of splicing factors affect the AS profiles of target genes . Among the 27 differential splicing genes, some are likely involved in regulation of transcription and protein stability, such as Solyc01g008370, which encodes a 26S proteasome regulatory subunit. The UPF3 homolog Solyc10g044450 regulating nonsense-mediated decay of PTC transcripts also showed differential alternative splicing in early fruit growth. This implicates that alternative splicing of some genes involved in maintaining transcript and protein stability is regulated by early fruit development.
High throughput RNA-seq, which can produce millions of reads in a single experiment, has the potential to detect novel transcripts and provides an additional way to facilitate gene prediction. Using RNA-seq, a large number of NTRs unlinked to the annotated loci have discovered in rice [26, 49, 50], soybean , grape  and Arabidopsis [36, 53]. In this study, we detected 2650 new unique transcripts from 2507 chromosomal regions where no gene has been predicted previously. Consistent with the reported analysis conducted in grape , transcripts from more than half of these NTRs are likely lncRNAs, indicating that these putative lncRNAs may play important roles in regulation of early fruit growth in tomato. Furthermore, the remaining about 1000 protein coding genes not predicted previously will be a valuable resource for genomic and genetic analysis of fruit growth in tomato. Therefore, the newly identified NTRs in this study will also facilitate updating the tomato genome annotation in the future.
In this study, we detected 59.3 % of the expressed multi-exon genes in the tomato genome that underwent AS and found that IR is the most abundant AS type. Comparison of AS events in different tissues revealed that multi-exon genes produced more splice variants in early growth fruits than did in seedlings and flowers. We also discovered that for many genes, transcription was regulated at isoform level rather than at gene level during early fruit growth. In addition, the identification of the more than two thousands of NTRs in this study provides a rich resource for future genomic and genetic analysis in tomato.
The wild relative of cultivated tomato S.pimpinellifolium LA1589 and the two cultivate tomatoes S.lycopersicum cv. Heinz1706 (LA4345) and LA2397 were obtained from the Tomato Genetics Resource Center at University of California, USA. Plants were grown in phytotrons at 20–25 °C under a humidity of 70–80 % and with daily illumination (150 mE · m−2 · s−1) for 16 h. Plants were fertilized weekly with all-purpose fertilizer and watered as needed.
RNA sequencing, read mapping and transcript assembly
Total RNA was extracted by Trizol reagent (Thermo Fisher Scientific, USA) from 7-days-old seedlings, anthesis flowers, fruits at 2, 5 and 10 dpa based on the methods described previously . Paired-end sequencing libraries were created using TruSeq stranded mRNA kit (RS-122-2101, Illumina Inc. USA) and sequenced on Illumina’s Miseq system using 500-cycles Miseq reagent kit (MS-102-2003, Illumina Inc. USA).
Because the cultivated tomato and its closest wild relative S.pimpinellifolium LA1589 have only 0.6 % nucleotide divergence in genome sequences , reads from LA1589 can be readily mapped to the reference Heinz1706 (S.lycopersicum) genome. The 7-days-old seedlings from LA1589 and Heinz1706 were included to make an overall assessment of the impact on AS detection by mapping of the LA1589 reads to the reference genome because of the comparability between the two samples. Therefore, all the 250 bp paired-end reads obtained in this study were mapped to reference genome (version ITAG2.5) using Tophat program v.2.0.12, guided by its corresponding annotation . Following parameters were used: −-read-mismatches 5—read-gap-length 3—read-edit-dist 5—library-type = fr-firststrand—splice-mismatches 0 and GTF. Mapped reads were then assembled by the Cufflinks program (version 2.2.1) using parameters: −GTF-guide –frag-bias-correct –min-frags-per-transfrag 10 . Then, differentially expressed genes at gene level (adjusted p value of 0.05 or less) were identified by comparisons between two consecutive stages of early fruit growth using the Cuffdiff tool with parameters used as follows: −frag-bias-correct –multi-read-correct –min-alignment-count 10 –FDR 0.05. The Cuffdiff program also reported genes showing differential splicing and estimated expression values at gene level. The abundance of individual mRNA isoforms was estimated by Cuffnorm using the same parameters as used in Cuffdiff.
NTRs were determined by comparing the assembled transcripts from all mapped reads to the annotated gene regions using the Cuffcompare program with parameters –r –s. New transcript sequences were then extracted and further assembled by Sequencher software (Gene Code Inc. USA) using gapped alignment. Cuffdiff, Cuffnorm and Cuffcompare are parts of the Cufflinks program.
Identification of AS events
AS events were predicted by the ASTALAVISTA program on the web server (http://genome.crg.es/astalavista/)  using the GTF files generated by Cufflinks. The AS events were classed into five types including the four basic types (AA, AD, ES, IR) and others that contain more than one of the four basic types. The output from ASTALAVISTA can be found in the supplementary materials (Additional file 7: “AS landscape.txt”).
GO ontology enrichment analysis of differentially expressed splice variants
Singular enrichment analysis (SEA) of GO ontology was conducted on differentially expressed genes at gene or isoform level using the online tools AgriGO (http://bioinfo.cau.edu.cn/agriGO/analysis.php). The SEA analysis was performed with statistical test of hypergeometric and multi-test correction by Bonferroni method. Over-represented functional categories of GO terms were selected for those with false discovery rate (FDR) smaller than 0.05.
RT-PCR validation of AS events and NTRs
Total RNA was extracted by Trizol reagent (Thermo Fisher Scientific, USA) from anthesis ovaries and 20 dpa fruits in addition to the tissues used for RNA-seq based on the methods described previously . After genomic DNA in these RNA samples was removed by RNase-free DNase according to the manufacturer’s protocol (New England BioLabs, USA), the total RNA (1 μg per sample) was used to synthesize first strand complementary DNA using the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). Ten percent of the reverse transcription products were subjected to PCR analysis. 30 and 35 cycles with annealing temperature at 55 °C for 45 s were used in RT-PCR validation of NTRs and AS events, respectively. Primer information can be found in Additional file 8: Table S5.
Availability data and materials
All raw reads obtained in this study have been deposited in the NCBI Short Read Archive under Bioproject PRJNA295119.
The work was supported by grants from Ministry of Science and Technology of People’s Republic of China (2012CB113900 and 2012AA100105) and Chinese Academy of Sciences (KSCX2-EW-J-12).
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