**3. Discussion**

Emerging evidence shows that ncRNAs play important roles in cellular functions and especially in biotic and abiotic stresses [12]. Among all the ncRNAs, miRNAs, which perform their functions by mRNA slicing or inhibition at the post-transcriptional level, were most intensively studied [34,35]. Unlike miRNAs, the regulatory function of lncRNAs is difficult to understand because of its complexity, since lncRNAs can fold into secondary or higher orders of structure that make them more flexible in targeting proteins or gene sites [36]. Although thousands of circRNAs have been identified, their functions are largely unknown, but their spatio-temporal expression and tissue specificity indicate their potential biological roles in plants [37,38]. The cross-talk among mRNAs, lncRNA, and circRNA mediated by MREs, regulates biological processes and produces mass regulatory networks [35]. To explore the regulatory functions and complex interactions of ncRNAs in chilling injury, deep sequencing and bioinformatics technology were employed. In total, 380 lncRNAs, 36 circRNAs, 18 miRNAs, and 4128 differentially expressed mRNAs were identified, and three comprehensive ceRNA networks were found, which indicated their specific regulatory roles in chilling injury in bell pepper.

The study of ncRNAs in bell pepper is presently scanty. Few studies were focused on the regulation of lncRNAs and miRNAs in fruit development and quality in hot and black pepper [26,28,39]. At the present time, studies of ncRNA regulation in chilling injury in bell pepper are limited to the field of mRNAs [29]. This is the first report on the differential expression of lncRNA, mRNA, circRNA, and miRNA in chilling injury in bell pepper. In addition, we finely identified 9764 novel lncRNAs, 213 novel circRNAs, and 161 novel miRNAs which were enriched the ncRNAs library. Furthermore, 380 differentially expressed lncRNAs, 36 circRNAs, 18 miRNAs, and 4128 mRNAs were identified between the control and the chilling groups, which indicated their specific regulatory roles played in chilling injury.

In order to explore the potential regulatory functions of the ncRNAs differentially expressed between control and chilling injury groups, GO analysis was performed to further annotate the biological functions of the differentially expressed ncRNAs and their target genes. We noticed that a significant amount of GO terms of the differentially expressed ncRNAs genes was related to response to abiotic stimulus, signal transduction, hormone-mediated signaling pathway, and response to cold, and the molecular functions included protein kinase activity, ATPase, and protein serine/threonine kinase activity. This phenomenon is very intriguing, revealing the vital roles that ncRNAs play in chilling injury. In accordance with the results of the GO analysis, KEGG pathway analysis also revealed pathways related to RNA degradation, peroxisome, plant hormone signal transduction, and carbon metabolism, which indicated their specific functions in the chilling response. In addition, for the differentially expressed mRNAs, numerous mRNAs which encode key enzymes, including superoxide dismutase (SOD), polyphenol oxidase (PPO), and peroxidase (POD,) involved in the protection again oxidative damage by reactive oxygen species (ROS), were found in our results. SOD converts superoxide anion (O<sup>2</sup> <sup>−</sup>) to hydrogen peroxide (H2O2), which in turn is converted to water by Catalase (CAT) and POD [40]. In our results, *SOD*, *PPO*, and *POD* were significantly upregulated, consistently with previous results [5]. Furthermore, in this study, numerous transcription factors, such as the *ERFs*, *MYB*, *NAC*, and *WRKY*, were significantly upregulated by chilling stress, which was consistent with previous results [5].

Recently, circRNAs were proposed to harbor miRNAs and were discovered to be enriched with functional miRNA-binding sites [41]. So far, there has been no report on ceRNAs in bell pepper fruit. Here, we constructed a lncRNA–circRNA–mRNA ceRNA network for bell pepper chilling stress based on our deep-sequencing data for the first time. In total, 162 mRNAs, 81 lncRNAs and 4 circRNAs were included in the ceRNA network. Several targets of the non-coding RNAs in the network were key enzymes in chilling injury, such as ATPase, which is an important enzyme in energy metabolism in bell pepper [42], serine/threonine protein kinase, and β-galactosidase, which are important in signaling and plant defense reaction and cell wall metabolism, respectively [37,43]. In addition, several transcription

factors, such as ethylene-responsive transcription factor and heat shock factors, which play specific regulatory roles in the chilling response, were identified [44,45]. In addition, a pathway network was also constructed with the key genes of the KEGG analysis, revealing that the most important pathway was involved in lipid transport and metabolism, which are important in the chilling stress process [46,47]. These findings provide a theoretical basis for deciphering novel mechanisms of chilling injury and for the functional characterization of ceRNA networks in the future studies.
