Identification and Characterization of Long Non-Coding RNAs: Implicating Insights into Their Regulatory Role in Kiwifruit Ripening and Softening during Low-Temperature Storage

Abstract

Long non-coding RNAs (lncRNAs) are crucial players regulating many biological processes in plants. However, limited knowledge is available regarding their roles in kiwifruit ripening and softening. In this study, using lncRNA-seq technology, 591 differentially expressed (DE) lncRNAs (DELs) and 3107 DE genes (DEGs) were identified from kiwifruit stored at 4 °C for 1, 2, and 3 weeks in comparison with non-treated control fruits. Of note, 645 DEGs were predicted to be targets of DELs (DEGTLs), including some DE protein-coding genes (such as β-amylase and pectinesterase). DEGTL-based GO enrichment analysis revealed that these genes were significantly enriched in cell wall modification and pectinesterase activity in 1 W vs. CK and 3 W vs. CK, which might be closely related to the fruit softening during low-temperature storage. Moreover, KEGG enrichment analysis revealed that DEGTLs were significantly associated with starch and sucrose metabolism. Our study revealed that lncRNAs play critical regulatory roles in kiwifruit ripening and softening under low-temperature storage, mainly by mediating the expression of starch and sucrose metabolism and cell wall modification related genes.

Keywords: fruit ripening and softening; kiwifruit; lncRNA-mRNA interaction; long non-coding RNA; low-temperature storage.

Figure 1

Physiological properties of kiwifruit during low temperature storage. (A–C) represent the change in firmness, vitamin C content, and soluble solid content during low-temperature storage, respectively. CK, 1 W, 2 W, and 3 W indicate that kiwifruit stored at 4 °C for 0, 1, 2, and 3 weeks, respectively. Different lowercases above curves represent a significant difference at p-value < 0.05 level.

Figure 2

Expression profiles of DEGs and DELs in kiwifruit during low-temperature storage. (A–C) represent volcanic map, Venn diagram, and heatmap of DEGs, respectively. (D–F) represent volcanic map, Venn diagram, and heatmap of DELs, respectively. Each dot in the volcanic map represents a gene, the red, green, and blue color represents the expression of this gene is up-regulated, down-regulated, and not changed, respectively. Each column in the heatmap represents a treatment, and each row represents a gene, the red color indicates high expression and blue color indicates low expression of a gene in this treatment.

Figure 3

Top 20 significantly enriched GO terms (p < 0.05) for DEGs identified in low-temperature stored kiwifruit. BP: biological process; CC: cellular component; MF: molecular function. (A) Significantly enriched GO terms by DEGs in the comparison of 1 W vs. CK. (B) GO terms significantly enriched by DEGs in the comparison of 2 W vs. CK. (C) GO terms significantly enriched by DEGs in the comparison of 3 W vs. CK.

Figure 4

KEGG pathway enrichment analysis of DEGs in low-temperature stored kiwifruit. (A) Top 20 pathways enriched by DEGs in the comparison of 1 W vs. CK. (B) Top 20 pathways enriched by DEGs in the comparison of 2 W vs. CK. (C) Top 20 pathways enriched by DEGs in the comparison of 3 W vs. CK.

Figure 5

Venn diagrams of DEGTLs. DEGs: differentially expressed genes; TGDELs: target genes of DELs. Overlapping sections indicate these DEGs were predicted as DEGTLs.

Figure 6

KEGG pathway enrichment analysis of DEGTLs in low-temperature stored kiwifruit. (A) Top 20 pathways enriched by DEGTLs in the comparison of 1 W vs. CK. (B) Top 20 pathways enriched by DEGTLs in the comparison of 2 W vs. CK. (C) Top 20 pathways enriched by DEGTLs in the comparison of 3 W vs. CK.

Figure 7

Expression analysis of selected genes and lncRNAs using qRT-PCR. The qRT-PCR results are shown in columns and the lncRNA-seq results are shown as curves. Different letters above columns represent a significant difference at p-value <0.05 level. CK, 1 W, 2 W, and 3 W indicate that kiwifruit stored at 4 °C for 0, 1, 2, and 3 weeks, respectively. Acc28118: Beta-amylase, Acc06893: Zinc finger protein, Acc20977: Beta-amyrin, Acc17513: Cytochrome b561 and DOMON domain-containing protein, Acc08288: NAC, Acc29506: Zinc transporter.