The phytohormone ethylene is well known for its important role in the ripening of climacteric fruit, such as tomato (Solanum lycopersicum). However, the role and mode of action of other plant hormones in climacteric fruit ripening regulation are not fully understood. Here, we showed that exogenous GA treatment or increasing endogenous gibberellin content by overexpressing the gibberellin synthesis gene SlGA3ox2 specifically in fruit tissues delayed tomato fruit ripening, whereas treatment with the GA biosynthesis inhibitor paclobutrazol (PAC) accelerated fruit ripening. Moreover, exogenous ethylene treatment cannot completely reverse the delayed fruit ripening phenotype. Furthermore, exogenous GA treatment of ethylene signalling mutant Never ripe (Nr) or SlEBF3-overexpressing lines still delayed fruit ripening, suggesting that GA involved in fruit ripening partially depends on ethylene. Transcriptome profiling showed that gibberellin affect the ripening of fruits by modulating the metabolism and signal transduction of multiple plant hormones, such as auxin and abscisic acid, in addition to ethylene. Overall, the results of this study provide new insight into the regulation of gibberellin in fruit ripening through mediating multiple hormone signals.

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