Tomato is one of the most popular commercial vegetables; however, its fruit shows high susceptibility to cracking. Cracks can occur throughout the fruit development stage during the ripening and post-harvest period [43-44], which may cause serious economic losses. Different hypotheses have been presented to explain the occurrence of tomato fruit cracking. Previous studies have shown that rapid fruit swelling and fruit cracking are closely related . Irregular temperatures or watering, especially a shift from a lower temperature to a much higher temperature or from extremely dry to very humid conditions, will lead to rapid swelling. If the flesh grows faster than the pericarp and the skin is not strong enough, cracking can easily occur. Cell senescence and apoptosis also influence skin strength and water absorption, which can in turn affect fruit cracking. In addition, a large differential between day and night temperatures will lead to the accumulation of carbohydrates. Fruits with high levels of carbohydrates absorb more water, grow much faster and are more likely to crack. In general, fruit cracking is a complex problem involving a mixture of nature and nurture. Previous studies have suggested it is not a single gene but many genes that work together to regulate fruit cracking [22-23]. Liu’s research suggests that plants have gradually developed complex signalling pathways to cope with adverse environmental stimuli . That is, plants perceive different stress signals from the circumstances in which they occur and then integrate these signals and respond to these different stresses by modulating the expression of related genes. Is it possible that cracking is also regulated by a complex network?
LncRNAs regulate tomato fruit cracking by coordinating gene expression in the hormone-redox-cell wall network
LncRNAs play important roles in epigenetic regulation, cell cycle regulation and many other activities. Here, we identified several lncRNAs that are involved in fruit cracking. LncRNAs mainly act in cis or trans on protein-coding genes to achieve their regulatory function. The principle of cis action is that the function of lncRNAs is related to their neighbouring genes . Most lncRNAs are not annotated, and we do not know their functions. To predict the functions of these lncRNAs, we performed functional analysis of lncRNA-targeted mRNAs and constructed an lncRNA-mRNA network (Fig. 7; Additional file 6). The results showed that the mRNAs in the network (Fig. 7a) were mainly enriched in the ‘oxidation-reduction process’, ‘oxidoreductase activity’, ‘hormone metabolic process’, ‘response to hormone stimulus’, ‘catalytic activity’, ‘cell wall organization’ and ‘external encapsulating structure’ categories. We classified the target genes into four categories (cell wall polysaccharide metabolism, oxidation-reduction processes, hormones and others) based on their functions and amounts.
Some lncRNAs specifically target functional mRNAs, and we can assume that the lncRNAs perform similar functions to their target mRNAs. For example, many of the target genes of XLOC_010878, XLOC_016662, and XLOC_033910 (Fig. 7b) are enriched in categories such as ‘dioxygenase activity’, ‘oxidation-reduction process’ and ‘oxidoreductase activity’, so we predicted their gene function as ‘redox regulation’.
Some lncRNAs are targets of significantly differentially expressed mRNAs with various functions. For example, for XLOC_16662 (Fig. 7c), the target genes (Solyc07g026650.2, Solyc08g081000.2, Solyc03g031880.2,., etc.) are enriched in the 'oxidoreductase activity' and 'dioxygenase activity' terms. XLOC_16662 also has other target genes, such as Solyc08g008120.2 ('negative regulation of abscisic acid-mediated signalling pathway'), and Solyc08g081010.2 ('cell wall thickening'). Previous studies have shown that redox, hormone and cell wall terms are all very important factors that can influence fruit cracking, so we speculate that lncRNA XLOC_16662 may play an important role in regulating tomato fruit cracking, along with XLOC_008464, XLOC_033910, XLOC_007053, XLOC_025351 and XLOC_040425.
Key genes regulating tomato fruit cracking
According to the gene expression analysis, 16 significantly differentially expressed genes are predicted to be related to fruit cracking in tomato, specifically genes such as Solyc07g026650.2, Solyc04g054830.2, Solyc07g017770.2, Solyc07g055990.2, Solyc04g072000.2, Solyc01g008710.2, etc. (Additional file 7). 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. 8a). For instance, the expression of Solyc12g011030.1, Solyc04g072000.2, Solyc09g075330.2, Solyc02g080530.2, Solyc07g055990.2 and Solyc09g008720.1 in the CR tomato showed a downward trend, while the expression in the CS tomato presented an upward trend. These genes encode proteins that function as pectin esterase, xyloglucan endotransglucosylase/hydrolase, and expansin, which play important roles in cell wall loosing and expansion and may also play a key regulatory role in tomato fruit cracking.
Finally, we mapped a pathway diagram (Fig. 8b) of fruit cracking based on these differentially expressed lncRNAs, mRNAs and previous studies [47-52]. Within this pathway, Solyc09g008720.1 (ethylene), Solyc02g080530.2 (peroxide) and Solyc09g075330.2 (pectinase) play important roles. Previous research suggests that ethylene influences fruit development and ripening (regulating cell wall-related PG and EXP gene expression)  and promotes programmed cell death of epithelial cells under ROS signalling . Li et al.  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 . 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. Simultaneously, the increase of auxin can promote the accumulation of H2O2 and the elongation of cells . Furthermore, Rayle and Cleland  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. 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.
Cell wall polysaccharide metabolic
The DEG Solyc08g077910.2 encodes an Expansin-like protein that breaks down the hydrogen bonds between molecules in the cell wall macromolecular network to promote the depolymerization of the network, which can lead to relaxation of the cell wall . In this experiment, the expression level of Solyc08g077910.2 was increased significantly after 8 h of irrigation (log2 fold-change=7.13395) in CS tomato. The increased expression of the expansin-like gene can relax the cell wall and may influence fruit cracking.
Solyc07g055990.2 and Solyc12g011030.1 encode xyloglucan endotransglucosylase/hydrolases 7 and 9, respectively, which mediate the cleavage and polymerization of β-1,4-xyloglucan in the primary cell wall. Xyloglucan is usually fused to the cell wall, and its oligosaccharides determine tissue tension . Jan  found that OsXTH8 is involved in the cell wall modification process in rice and is highly expressed in the vascular bundle of the sheath and the young roots, in which the cells are rapidly elongated and differentiated. In addition, it can respond to gibberellin. He  found that OsXTH5, OsXTH19, OsXTH20, OsXTH24 and OsXTH28 play important roles in the elongation of rice peduncles and can respond to drought stress. These studies indicate that the OsXTH gene family plays an important role in the regulation of the structural function of rice cell walls. In this experiment, the expression levels of Solyc12g011030.1 and Solyc07g055990.2 in CS tomato showed an upward trend, while they showed a downward trend in the CR tomato (Fig. 8a). Simultaneously, the expression level in CS tomato was significantly higher than that in CR tomato. This illustrates that the CR tomato may exhibit a greater osmotic stress resistance ability with downregulation of the XTH gene that can strengthen the cell wall upon encountering water stress.
The DEG Solyc10g080210.1 (pectinase) can remove the methyl group from polygalacturonic acid; during tomato maturation, the degree of methylation decreases from 90% in the green ripening period to 35% in the red ripening period , which accelerates the degradation of the cell wall. In an antisense PaPG1 transgenic study of strawberry, the expression level of PG was significantly inhibited, and the degree of fruit softening was significantly delayed .
Previous studies have shown that peroxidase in the cell wall leads to cell wall sclerosis by causing cross-linking of cell wall components, thereby inhibiting cell elongation [59-61]. Peroxidase can also directly regulate plant cell elongation by controlling H2O2 levels . Solyc02g080530.2 encodes peroxide, whose levels are significantly higher in CS tomato than in CR tomato. The expression of these genes in CS tomato fruits may increase cell wall hardness and hinder the elongation of the cell wall, which will lead to fruit cracking when water absorption swelling occurs. Solyc01g081250.2 encodes glutathione-S-transferase (GST). The GST superfamily enzymes exhibit multiple functions in plants. They are not only involved in primary metabolism and secondary metabolism , but they can also protect plants from oxidative damage and heterogeneous substances [64-66]. According to the data analysis, the gene expression of Solyc01g081250.2 in the CR tomato was significantly higher than that in the CS tomato after 0 h, 8 h and 30 h of irrigation treatment. Higher expression of GST in CR tomato can better maintain cell vigour and be beneficial to tomato fruits when coupled with water stress.
Previous research has shown that hormones can regulate the expression of cell wall-related genes. Trainotti  studied the expression of 32 genes related to cell wall synthesis and degradation. Their research showed that the expression of these genes in unsoftened fruits can be inhibited by ethylene, while ethylene promotes the expression of these genes during fruit ripening and softening. At the same time, ethylene inhibits and promotes dual regulatory effects on the formation of plant secondary metabolites [68-70]; TAPG1, encoding a cell wall-degrading enzyme, can be induced by ethylene at the transcriptional level in tomato ; Rose  showed that ethylene regulates LeEXP1, which is specifically expressed only during fruit ripening. The pathway of ethylene biosynthesis in plants is the methionine cycle. In this study, KEGG functional analysis of DEGs revealed significant enrichment in the methionine metabolic pathway. Solyc11g042560.1 encodes an ethylene receptor, while Solyc09g008720.1 encodes an ethylene-responsive transcription factor, and their expression levels are significantly upregulated after irrigation and are higher in CS tomato than in CR tomato.