RNA sequencing and identification of lncRNA and mRNA
In total, we obtained 0.81 to 1.14 billion raw reads and 0.79 to 1.14 billion clean reads from CR and CS tomatoes at various time points (0 h, 8 h and 30 h of saturated irrigation treatment) (Table 1). Through genomic comparison, Cufflinks splicing, and CPC2 and PFAM analysis, we identified 1 annotated lncRNA, 2508 putative lncRNAs , 33784 annotated mRNAs and 409 novel mRNAs ( Additional file 3).
Feature analysis of lncRNAs and identification of lncRNA-mRNA pairs
The average length of the obtained lncRNAs was 1470 nt, which was similar to that of the mRNAs (1221 nt); the average number of exons and average ORF length of the identified lncRNAs were 2.6 and 88.5 bp, which were much lower values than those for the mRNAs (4.7 and 347 bp) (Fig. 3), consistent with previous studies [55, 56 ]. At the same time, we used phyloP to separately score the lncRNAs and mRNAs, and the sequence conservation of the lncRNAs was lower than that of mRNAs, which is consistent with previous studies [57]. We identified 21048 lncRNA-mRNA pairs with target relationships upstream and downstream of 2508 lncRNAs (Additional file 4).
Differential expression analysis
Differentially expressed mRNAs and lncRNAs were analysed in CR and CS tomatoes by using edge R software (Fig. 4) and the number of differentially expressed genes was listed in the Additional file 5. mRNAs and lncRNAs with a Q-value<0.05 and |log2 fold-change|> 1 were selected as differentially expressed genes.
Functional prediction of DEGs
To investigate the trends in gene functions and enrichment for DEGs, we performed GO (Gene Ontology) analysis of the selected mRNAs (Fig. 5; Additional file 6). The results showed that DEGs in the CR tomato were involved in a series of biological processes, such as regulation of biological process, biological regulation and regulation of cellular process, as well as catalytic activity. For the CS tomato, DEGs were mainly involved in single-organism metabolic process, biological process and catalytic activity. Between the CR and CS tomatoes, before irrigation treatment (0 h), DEGs were significantly enriched in oxidoreductase activity; after 8 h of irrigation treatment, there were some DEGs enriched in fruit ripening, anatomical structure maturation, and ageing; after 30 h of irrigation treatment, the number of DEGs enriched in the catalytic category was the highest, followed by the single-organism metabolic process and oxidation-reduction process categories, and cell components.
To better understand the function of the DEGs, significantly enriched KEGG pathways were analysed (Fig. 6; Additional file 7). The results showed that the DEGs were mainly enriched in the ‘biosynthesis of secondary metabolites’, ‘cysteine and methionine metabolism’, ‘metabolic pathways’, ‘plant-pathogen interaction’, ‘photosynthesis-antenna protein’, ‘photosynthesis’, ‘histidine metabolism’ and ‘circadian rhythm-plant’ categories.
DEGs (cell wall, redox, hormone related) regulate tomato fruit cracking
According to the gene expression analysis, 16 significantly differentially expressed genes (Additional file 8) were predicted to be related to fruit cracking in tomato, such as Solyc02g080530.3 (Peroxide, POD), Solyc01g008710.3 (Mannan endo-1,4-beta-mannosidase, MAN), Solyc08g077910.3 (Expanded, EXP), Solyc09g075330.3 (Pectinesterase, PE), Solyc07g055990.3 (Xyloglucan endotransglucosylase-hydrolase 7, XTH7), Solyc12g011030.2 (Xyloglucan endotransglucosylase-hydrolase 9, XTH9), Solyc10g080210.2 (Polygalacturonase-2, PG2), Solyc08g081010.2 (Gamma-glutamylcysteine synthetase, gamma-GCS), Solyc09g008720.2 (Ethylene receptor, ER), Solyc11g042560.2 (Ethylene-responsive transcription factor 4, ERF4) etc. (Table. 2 [58-81] ). Hierarchical clustering analysis showed that the expression trends or levels of these genes in the two varieties were completely different after the irrigation treatment (Fig. 7a). For instance, the expression of XTH7, XTH9, PE and POD in the CR tomato showed a downward trend, while the expression in the CS tomato presented an upward trend. These genes play important roles in cell wall loosing and expansion. As disassembly of the fruit cell wall can influence fruit cracking [30], These plant cell-wall loosing genes may also play a key regulatory role in tomato fruit cracking. At the same time, we used Tomato Gene Expression Atlas (http://tea.solgenomics.net/expression_viewer/input) to verify the gene expression, and found that most differentially expressed genes in this experiment were expressed in tomato pericarp in red ripe stage. Among them, GCS, MAN and PG, the antioxidative genes and cell-wall degrading enzyme-associated genes showed the highest expression. Whereas high mRNA levels were never present in ERF. These might be because that the ERF is an upstream regulator [82], so it never presented a high expression in red ripe stage. The differences in gene expression in this experiment and Tomato Gene Expression Atlas might be due to different varieties, treatment and detection standards (Fig. 7b).
Finally, we mapped a pathway diagram (Fig. 7c) of fruit cracking based on these differentially expressed lncRNAs, mRNAs and previous studies [82-88]. Within this pathway, ERF, POD, PG and PE play important roles. Previous researches suggests that ethylene influences fruit development and ripening (regulating cell wall-related PG and EXP gene expression) [82] and promotes programmed cell death of epithelial cells under ROS signalling [83]. Li et al. [84] showed that ARFs represent a point of cross-talk between ethylene and auxin signalling. Furthermore, auxin induces the production of ROS, and H2O2 decomposes polymers at the cell wall by producing ·OH [85]. Programmed cell death leads to a reduction in or loss of permeability of the plasma membrane, which in turn influences fruit cell activity, water absorption and cracking [86]. Simultaneously, the increase of auxin can promote the accumulation of H2O2 and the elongation of cells [87]. Furthermore, Rayle and Cleland [88] proposed the acid growth theory indicating that hydrogen ions may exert a purely chemical or physical effect, such as cleavage of acid-labile bonds on the wall, or they may activate normal enzymatic processes directly or indirectly, potentially leading to wall loosening. We analyzed the regulatory element in the promoter sequence of PG, PE, EXP and XTH7 and found that there are Ethylene-responsive element and auxin-responsive element. Based on these findings, we speculate that the regulatory network of fruit cracking, especially the coexpression of cell wall-, redox-, and hormone-related mRNAs and their corresponding lncRNAs, influences fruit cracking.
qRT-PCR validation of DEGs
Genes showing upregulated and the downregulated expression were randomly selected from the DEGs for qRT-PCR verification. The results of qRT-PCR revealed that most of these mRNAs shared similar expression tendencies to those indicated by the mRNA-Seq data, which can validate the reliability of our sequence data and our research results from the present study (Additional file 9 ). The expression levels detected by the two methods were slightly different, which might have been due to the different detection ranges and sensitivities of the two detection methods. The comparison of the relative expression measured by qRT-PCR and RNA-seq was showed in Additional file 10, R2>0.7 confirmed the reliability of the RNA-Seq analysis results.