A competitive endogenous RNA network involving <i>RiLinc6978</i> and miR9474-5p regulates fruit softening in tomato

Fruit softening determines fruit quality and influences postharvest storage and transport. Pectin degradation is mediated by a series of cell wall-modifying enzymes and orchestrates cell wall breakdown during ripening. Herein, we identified a competitive endogenous RNA (ceRNA) network that regulates pectin metabolism in tomato (Solanum lycopersicum). This ceRNA network comprises the long, intergenic, non-coding RNA (lincRNA) Ripening-induced lincRNA 6978 (RiLinc6978), the microRNA (miRNA) miR9474-5p, and multiple pectin-degrading enzymes. RiLinc6978 exhibits upregulated abundance concomitant with fruit ripening. Transcriptomic analysis and functional validation showed that RiLinc6978 promotes pectin demethylation and degradation, thereby accelerating fruit ripening and softening. Conversely, miR9474-5p suppresses these processes, delaying softening and prolonging ripening. RiLinc6978 and miR9474-5p are localized to the cytoplasm and the nucleus. Biotinylated miRNA pull-down assay revealed that miR9474-5p associates with multiple transcripts encoding pectin-degrading enzymes. 5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE) further validated miR9474-5p-directed cleavage of a transcript encoding pectin methylesterase, suggesting that miR9474-5p contributes to pectin degradation via post-transcriptional suppression of pectin-degrading enzymes. Furthermore, RiLinc6978 and miR9474-5p interact, with RiLinc6978 functioning as a molecular sponge to attenuate miR9474-5p-mediated target gene silencing. Our study elucidates a ceRNA regulatory module capable of fine-tuning pectin metabolism and softening dynamics, thereby providing a molecular framework for targeted breeding strategies aimed at optimizing tomato fruit quality.