Tomato is one of the most popular commercial vegetables, however, its fruit has 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 researches show that rapid fruit swelling and fruit cracking are closely related . Irregular temperature or watering, especially going from lower temperature to very higher, or extremely dry to very humid conditions, will lead to a rapidly swelling. If the flesh grows faster than the pericarp and the skin is not strong enough, cracking can easily occures; Besides, cell senescence and apoptosis will influence skin strength and water absorbing, this in turn can affect fruit cracking; In addition, a large differential between day and night temperature will lead to the accumulation of carbohydrate. Fruits with lots of carbohydrate absorb more water, will grow large much sooner and are more likely to cracking. In general, fruit cracking is a complex problem. It is a mixture of nature and nurture. Previous studies have suggest that there is no single gene, but many genes work together to regulate fruit cracking [22-23]. Liu’s research suggests that plants have been gradually developed a complex signal pathway to cope with adverse environmental stimulations . That is, plants perceive different stress signals from circumstances, to integrate them and to respond to these different stresses by modulating the expression of related genes, plant perceives and responds to environmental stimulations. Is fruits cracking regulated by a complex network too?
LncRNA regulates tomato fruit cracking by coordinating gene expression in hormone-redox-cell wall network
LncRNA plays important roles in epigenetic regulation, cell cycle regulation and many other activities. Here, we identified several lncRNAs that are involved in fruit cracking. LncRNA mainly functions by cis or trans on protein-coding genes to achieve its regulatory function. The principle of cis action is that the function of lncRNA is related to its neighboring gene . Most lncRNAs are not annotated, and we can't know their function. In order to predict the function of these lncRNAs, we performed functional analysis of lncRNA-targeted mRNAs, and constructed lncRNA-mRNA network (Fig. 7, Additional file 6). The results showed that the mRNAs in the network (Fig. 7a) are mainly enriched in ‘oxidation-reduction process’, ‘oxidoreductase activity’, ‘hormone metabolic process’, ‘response to hormone stimulus’, ‘catalytic activity’, ‘cell wall organization’ and ‘external encapsulating structure’. And we classify the target genes into four categories (cell wall polysaccharide metabolic, oxidation-reduction processe hormone and others) based on their function and amount..
Some lncRNAs are target for specifically functional mRNAs, we can assume that the lncRNAs perform the similar functions as their target mRNAs. For example, XLOC_010878, XLOC_016662, XLOC_033910 (Fig. 7b), many of their target genes are enriched in ‘dioxygenase activity’, ‘oxidation-reduction process’ and ‘oxidoreductase activity’ ect., so we predicted their gene function as “redox regulation”.
Some lncRNAs are targets for significantly differentially expressed mRNAs with various functions. Such as XLOC_16662 (Fig. 7c), its target genes (Solyc07g026650.2, Solyc08g081000.2, Solyc03g031880.2, ect.) are enriched in 'oxidoreductase activity' and 'dioxygenase activity' term, For the other target genes, Solyc08g008120.2 is enriched in 'negative regulation of abscisic acid mediated signaling pathway' term, and Solyc08g081010.2 is enriched in 'cell wall thickening', 'callose deposition in cell wall' and 'cellulose metabolic process' terms. Previous researches show that redox, hormone and cell wall are all very important factors that can influence fruit cracking, so we speculat that the lncRNA XLOC_16662 may play important role in regulating tomato fruit cracking. So did XLOC_008464, XLOC_033910, XLOC_007053, XLOC_025351 and XLOC_040425.
Key gene regulates tomato fruit cracking
According to the gene expressing analysis, GO and KEGG functional analysis, as well as the gene function annotated on the website (https://solgenomics.net), 16 significantly differentially expressed genes are predicted to related to fruit cracking on tomato, specifically Solyc07g026650.2, Solyc04g054830.2, Solyc07g017770.2, Solyc07g055990.2, Solyc04g072000.2, Solyc01g008710.2, ect (Additional file 7). Hierarchical clustering analysis shows that the expression trends or quantity of these genes in the two varieties are 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. The gene functions of these genes were pectin esterase, xyloglucan endotransglucosylase/ hydrolase, expansin, which play an important role 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 lncRNA, mRNA and previous studies [47-52], and also predicted key genes to play a key regulatory role in a certain pathway, such as Solyc09g008720.1, ethylene; Solyc02g080530.2, peroxide and Solyc09g075330.2, pectinase, ect . Previous research suggest that ethylene influences fruit development and ripening (regulating PG and EXP gene expression) , promotes programmed cell death of epithelial cells under ROS signaling . Li et al  shows that ARFs serve as a cross talk point between the ethylene and auxin signaling. Furthermore, auxin induces the production of ROS, and H2O2 decomposes the polymer on the cell wall by producing ·OH . And cell programmed death leads to reduced or lost permeability of the plasma membrane. This in turn influence fruit cell activity, water absorption and cracking. Simultaneously the increase of auxin can promote the accumulation of H2O2 and promote the elongation of cells . Furthermore, Rayle and Cleland  proposed the acid growth theory. Which means on one hand, hydrogen ion exerts a purely chemical or physical effect, such as cleavage of some acid-labile bonds on the wall. Alternatively, it may activate the normal enzymatic processes directly or indirectly, which may lead to wall loosening. Based on these, we speculate the regulatory network of fruit cracking. And coexpression of cell wall, redox, hormone related mRNAs and its corresponding lncRNA influence fruit cracking.
Cell wall polysaccharide metabolic
The DEG Solyc08g077910.2 encodes an Expansin-like protein according to the gene function annotation of the tomato website. It breaks down the hydrogen bonds between its molecules to promote the depolymerization of the cell wall macromolecular network, which can lead to the relaxtion of the cell wall . In this experiment, the expression level of Solyc08g077910.2 increased significantly at 8h of irrigation (log2(fold change)=7.13395) in CS tomato. The increased expression of expansin-like gene can relax the cell wall and that may influence fruit cracking.
Solyc07g055990.2 (Xyloglucan endotransglucosylase/ hydrolase7), Solyc12g011030.1 (xyloglucan endotransglucosylase/ hydrolase9) encodes a class of xyloglucan endotransglucosylase/hydrolase, which mediates the cleavage and polymerization of β-1,4-xyloglucan in the primary cell wall and is thought to play an important role in the formation and remodeling of xyloglucan. Xyloglucan usually fused in the cell wall, xyloglucan 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 vigorously elongated and differentiated, while it can respond to gibberellin, involved in the cell extension process. He  found that OsXTH5, OsXTH19, OsXTH20, OsXTH24 and OsXTH28 play important roles in the elongation of rice peduncle 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, which showed a downward trend in the CR tomato (Fig. 8a), and the expression level in CS tomato was significantly higher than that in the CR tomato. This illustrates that the CR tomato may have a possibly higher osmotic stress resistance ability with a down-regulation of the XTH gene that can strengthen the cell wall upon encountering the water stress.
Solyc10g080210.1 encodes a polygalacturonase whose main function is to hydrolyze α-1,4 glycosidic bonds in the cell wall polygalacturonic acid, polygalacturonic acid is a component of pectin, so it is also called pectinase . Polygalacturonic acid exists in the cell wall in a highly methylated form, and the methyl group is removed from the pectin of the cell wall by the pectin methylesterase during tomato maturation, the degree of methylation decreased from 90% in the green ripening period to 35% in the red ripening period , which accelerated the degradation of the cell wall. In the antisense PaPG1 transgenic study of strawberry, the expression level of PG was significantly inhibited, and the degree of fruit softening was also 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 [60-62]. Peroxidase can also directly regulates plant cell elongation by controlling H2O2 levels . Solyc02g080530.2 encodes peroxide, which is significantly higher in CS tomato than in CR tomato. The expression of these gene may enhances the cell wall hardness and hinders the elongation of the cells of cell wall, resulting in the cracking of the fruit response to water swelling after the irrigation treatment. Solyc01g081250.2 encodes Glutathione-S-transferase(GST). GST is a super-family enzyme with multiple functions in plants. It is not only involved in primary metabolism and secondary metabolism , it can also protect plants from oxidative damage and heterogeneous substances [65-67]. According to the data analysis, the gene expression of Solyc01g081250.2 in the CR tomato was significantly higher than that of CS tomato at the 0h, 8h and 30h treated with irrigation. The higher expression of GST in CR tomato can better maintain cell vigor and be beneficial to tomato fruits when coupling with water stress.
Previous research showed that hormone can regulate the expression of cell wall related genes. Trainotti  studied the expression of 32 genes in the development and softening of peach fruit, which are related to cell wall synthesis and degradation. 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; Ethylene inhibits and promotes the dual regulation effect on the formation of plant secondary metabolites, which is the result of the interaction of ethylene with various factors inside and outside the cell [69-71]; TAPG1, the cell wall degrading enzyme gene, can be induced by ethylene at the transcriptional level in tomato; Rose  identified 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 are significantly enriched in the methionine metabolic pathway. Solyc11g042560.1 encodes ethylene receptor, Solyc09g008720.1 is ethylene-responsive transcription factor, their expression levels are significantly up-regulated after irrigation and higher in CS tomato than in CR tomato.
In this study, only two biological replicates were used, and there may be a certain false positive rate. Therefore, we randomly selected 9 DEGs for real-time PCR, which proved to be consistent with high-throughput sequencing. It is indicated that the differential genes obtained by high-throughput sequencing are reliable.