**4. Discussion**

Although genome-wide lncRNA searches and related studies have been carried out in *Arabidopsis thaliana* [60], rice [61], maize [62], wheat [63], and *Populus trichocarpa*, identification of lncRNA functions in plant response to allelopathy is lacking. In this study, an RNA-seq experiment was carried out on tomato plants treated with different concentrations of PHBA, which systematically identified plant allelopathy-related lncRNAs. The tomato material we used in this study is a new variety that is widely planted in North China and thus has an important reference value for plant allelopathy in vegetable production. In addition, the differentially expressed lncRNAs under different PHBA treatments were valuable for studying plant allelopathy in *S. lycopersicum*. Moreover, as an important vegetable crop grown worldwide, *S. lycopersicum* is considered a reference species for *Solanum* genomic studies [64]. The genome-wide characterization of lncRNAs in *S. lycopersicum* will provide a unique annotation resource for the *S. lycopersicum* genome.

Gene expression can be cis- or trans-regulated by lncRNAs [65]. Cis-acting lncRNAs control gene expression around their transcription sites, while trans-acting lncRNAs regulate the expression of genes at independent loci [66–68]. Additionally, the overlap of some cis- and trans-acting lncRNAs increases the complexity of the molecular roles of lncR-NAs [50,69]. Since lncRNAs are often coexpressed with their TPGs, a method for inferring the putative biological functions of lncRNAs was developed based on the relationships between the expressions of lncRNAs and TPGs [50,70]. The CTPGs and TTPGs of lncRNAs

were predicted, and a coexpression analysis was performed [50]. Strong coexpression between cis- or trans-acting lncRNAs and their TPGs was found in this study. To date, no lncRNA has been characterized in plant allelopathy processes. Further investigation of these lncRNAs that are highly coexpressed with TPGs with known functions may provide some clues that will enable discovery of the functions of lncRNAs in gene expression regulation of allelopathy.

Compared with the study of functionality, the study of evolution needs to be further explored. For plant lncRNAs, a recent BLAST search of *B. rapa* lncRNAs against the lncRNAs of *Arabidopsis thaliana* yielded few homologs (approximately 5%) [50], indicating that evolutionary conservation is limited in plant lncRNAs [50]. In this study, we found that only approximately 0.15% (98 out of 65,178) of lncRNAs in *S. lycopersicum* had homologs in *Arabidopsis thaliana*. This result also suggested limited evolutionary conservation in plant lncRNAs.

Recent studies have found lncRNAs involved in miRNA regulation mechanisms as ceRNAs, which were initially studied in *Arabidopsis thaliana* and tobacco [71,72]. In this study, we predicted the relationships between lncRNAs, miRNAs, and mRNAs using the miRNAs that were previously identified in *S. lycopersicum*. We propose that certain interactions between these lncRNAs, mRNAs, and miRNAs play fundamental roles in plant allelopathy.

#### **5. Conclusions**

In conclusion, screening and functional analysis of the genome of *S. lycopersicum* enabled the identification of a set of lncRNAs. The analysis of their functional links with mRNAs and miRNAs and the similarity alignment and conservation analyses of lncRNAs in *S. lycopersicum* and *Arabidopsis thaliana* may provide some molecular clues about plant allelopathy due to PHBA in *S. lycopersicum*.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/horticulturae8121134/s1, Data S1: The coexpression relationships between lncRNAs and protein-coding gene pairs, Figure S1: Informatics pipeline for the identification of lncRNAs in *S. lycopersicum*, Figure S2: Experimental validation of the lncRNA expression by qRT-PCR. (The horizontal axis represents different treatments of PHBA, the vertical axis represents the relative expression level of lncRNAs.), Table S1: The primers used in this study, Table S2: LncRNAs identified in *S. lycopersicum*, Table S3: Numbers of lncRNAs and PGs detected in *S. lycopersicum*, Table S4: Differential expressions of lncRNAs and mRNAs, Table S5: LncRNAs and their CTPGs, Table S6: The weighted gene co-expression network analysis modules of lncRNAs and corresponding TPGs, Table S7: GO terms and annotations of cis-regulated modules, Table S8: LncRNAs and cis-regulated target mRNAs that have correlation coefficients greater than 0.9 and *p*-values less than 1.0 <sup>×</sup> <sup>10</sup><sup>−</sup>5, Table S9: Cis-regulated target mRNAs that are differentially expressed, Table S10: LncRNAs and their trans-regulated corresponding target mRNAs analyzed using LncTar, Table S11: GO terms and annotations of trans-regulated modules, Table S12: LncRNAs and trans-regulated target mRNAs that have correlation coefficients greater than 0.9 and *<sup>p</sup>*-values less than 1.0 <sup>×</sup> <sup>10</sup>−5, Table S13: Trans-regulated target mRNAs that are differentially expressed, Table S14: Similarity alignment of lncRNAs in *S. lycopersicum* and *Arabidopsis thaliana*, Table S15: MiRNA target prediction by psRNATarget, Table S16: The lncRNA-miRNA-mRNA pairs with weight values greater than 0.15.

**Author Contributions:** Conceptualization, G.L.; Formal analysis, G.L.; Funding acquisition, J.G.; Software, G.L.; Validation, Y.N.; Writing—original draft, G.L.; Writing—review & editing, J.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was founded by the National Natural Science Foundation of China, grant numbers: 31770706 and 32071789.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The RNA sequencing read data have been deposited in the Short Read Archive (SRA) data library (accession number: SRP239079).

**Conflicts of Interest:** The authors declare no conflict of interest.
