Key transcription factors regulate fruit ripening and metabolite accumulation in tomato Huimin Jia, Yaping Xu, Yuanwei Deng, Yinhuan Xie, Zhongshan Gao, Zhaobo Lang, Qingfeng Niu Abstract: Fruit ripening is a complex process involving dynamic changes to metabolites and is controlled by multiple factors, including transcription factors (TFs). Several TFs are reportedly essential regulators of tomato (Solanum lycopersicum) fruit ripening. To evaluate the effects of specific TFs on metabolite accumulation during fruit ripening, we combined CRISPR/Cas9-mediated mutagenesis with metabolome and transcriptome analyses to explore regulatory mechanisms. Specifically, we generated various genetically engineered tomato lines that differed regarding metabolite contents and fruit colors. The metabolite and transcript profiles indicated that the selected TFs have distinct functions that control fruit metabolite contents, especially carotenoids and sugars. Moreover, a mutation to ELONGATED HYPOCOTYL5 (HY5) increased tomato fruit fructose and glucose contents by approximately 20% (relative to the wild-type levels). Our in vitro assay showed that HY5 can bind directly to the G-box cis-element in the Sugars Will Eventually be Exported Transporter (SWEET12c) promoter to activate expression, thereby modulating sugar transport. Our findings provide insights into the mechanisms regulating tomato fruit ripening and metabolic networks, providing the theoretical basis for breeding horticultural crops that produce fruit with diverse flavors and colors. Introduction: Tomato is one of the most important vegetable crops worldwide. It is also a model plant for investigating climacteric fruit ripening because of its short life cycle and relatively small genome. The aroma-related volatiles, fruit flavors, and characteristic pigments typically produced during the fruit ripening period are important for breeding and genetic selection. Plant metabolites, such as carotenoids, flavonoids, and sugars, undergo dramatic changes during the softening of fruit in the ripening transition stage, making them important indicators of fruit maturity and quality. The metabolic changes that occur as tomato fruit ripen are controlled by phytohormones (mainly ethylene) and numerous ripening-related genes (RRGs). Over the past 40 years, there have been considerable advances in tomato genetics research. Results and discussion: Ripening-related TFs alter the tomato fruit carotenoid content via pathways dependent or independent of ethylene. The functional classification of the targeted TFs revealed they affect many biological processes associated with fruit ripening. The expression of carotenoid and ABA accumulation-related genes is mediated by ripening-related TFs in tomato. Metabolome analyses clarified the effects of these TFs on fruit ripening. Transcriptional regulation of the metabolite biosynthesis genes in tomato. Conclusions: In this study, the CRISPR/Cas9 system was used to generate mutants. TheKey transcription factors regulate fruit ripening and metabolite accumulation in tomato Huimin Jia, Yaping Xu, Yuanwei Deng, Yinhuan Xie, Zhongshan Gao, Zhaobo Lang, Qingfeng Niu Abstract: Fruit ripening is a complex process involving dynamic changes to metabolites and is controlled by multiple factors, including transcription factors (TFs). Several TFs are reportedly essential regulators of tomato (Solanum lycopersicum) fruit ripening. To evaluate the effects of specific TFs on metabolite accumulation during fruit ripening, we combined CRISPR/Cas9-mediated mutagenesis with metabolome and transcriptome analyses to explore regulatory mechanisms. Specifically, we generated various genetically engineered tomato lines that differed regarding metabolite contents and fruit colors. The metabolite and transcript profiles indicated that the selected TFs have distinct functions that control fruit metabolite contents, especially carotenoids and sugars. Moreover, a mutation to ELONGATED HYPOCOTYL5 (HY5) increased tomato fruit fructose and glucose contents by approximately 20% (relative to the wild-type levels). Our in vitro assay showed that HY5 can bind directly to the G-box cis-element in the Sugars Will Eventually be Exported Transporter (SWEET12c) promoter to activate expression, thereby modulating sugar transport. Our findings provide insights into the mechanisms regulating tomato fruit ripening and metabolic networks, providing the theoretical basis for breeding horticultural crops that produce fruit with diverse flavors and colors. Introduction: Tomato is one of the most important vegetable crops worldwide. It is also a model plant for investigating climacteric fruit ripening because of its short life cycle and relatively small genome. The aroma-related volatiles, fruit flavors, and characteristic pigments typically produced during the fruit ripening period are important for breeding and genetic selection. Plant metabolites, such as carotenoids, flavonoids, and sugars, undergo dramatic changes during the softening of fruit in the ripening transition stage, making them important indicators of fruit maturity and quality. The metabolic changes that occur as tomato fruit ripen are controlled by phytohormones (mainly ethylene) and numerous ripening-related genes (RRGs). Over the past 40 years, there have been considerable advances in tomato genetics research. Results and discussion: Ripening-related TFs alter the tomato fruit carotenoid content via pathways dependent or independent of ethylene. The functional classification of the targeted TFs revealed they affect many biological processes associated with fruit ripening. The expression of carotenoid and ABA accumulation-related genes is mediated by ripening-related TFs in tomato. Metabolome analyses clarified the effects of these TFs on fruit ripening. Transcriptional regulation of the metabolite biosynthesis genes in tomato. Conclusions: In this study, the CRISPR/Cas9 system was used to generate mutants. The