Tomato MADS-RIN regulates GAME5 expression to promote non-bitter glycoalkaloid biosynthesis in fruit

A well-known defense-associated steroidal glycoalkaloid (SGA) metabolic shift eliminates the bitterness and toxicity of ripe tomato fruits. This study was conducted to clarify the effects of MADS-RIN (RIN) and its cofactors on SGA metabolism in tomato fruits. Using a CRISPR/Cas9-based gene-editing system, we mutated RIN and two cofactor genes (FUL1 and FUL2). The observed changes to fruit color and size in the mutants reflected the overlapping and distinct effects of RIN, FUL1, and FUL2 on fruit ripening. According to a UPLC-MS/MS analysis, the RIN and cofactor mutants had decreased levels of the relatively non-toxic metabolite esculeoside A, but they accumulated toxic SGA pathway intermediates, suggesting RIN and its cofactors are directly involved in esculeoside A biosynthesis. Transcriptome and qPCR analyses detected the downregulated expression of GAME5, which encodes a key enzyme mediating esculeoside A biosynthesis. ChIP-seq and ChIP-qPCR analyses confirmed GAME5 is targeted by RIN. RIN was observed to activate GAME5 transcription by binding to two non-canonical CArG-boxes in the GAME5 promoter. Additionally, RIN promotes SGA metabolism independently of ethylene. Collectively, these findings enhance our understanding of the molecular mechanism governing tomato fruit ripening and SGA biosynthesis. Furthermore, they may be useful for improving tomato fruit quality and safety.