SlAN2-like Orchestrates Anthocyanin Production and pH Homeostasis in Tomato via SlPH3 and SlMYB76

Anthocyanin accumulation and vacuolar pH are closely linked in fruit pigmentation and quality. This study demonstrates that the R2R3-MYB transcription factor SlAN2-like plays a dual role in tomato by enhancing anthocyanin biosynthesis and modulating fruit peel pH. Loss-of-function mutants of <i>SlAN2-like</i> in tomato cv. Indigo Rose showed reduced anthocyanins and a higher pH, indicating its involvement in pH homeostasis. Transcriptomics revealed <i>SlPH3</i>, a WRKY transcription factor, as a key downstream target coregulated by SlAN2-like. Overexpression of <i>SlPH3</i> in <i>Nicotiana benthamiana</i> enhanced vacuolar acidification, whereas the <i>SlPH3</i> knockout line in cv. Indigo Rose showed decreased anthocyanin content, higher pH, and downregulation of core biosynthetic genes. Additionally, SlMYB76 was identified as a repressor that binds the <i>SlPH3</i> promoter and suppresses its expression. SlAN2-like ionization activates <i>SlMYB76</i>, forming a feedback loop that balances anthocyanin production and pH control. These results uncover a hierarchical regulatory network with SlAN2-like coordinating pigmentation and vacuolar acidification via SlPH3 and SlMYB76 during tomato fruit development.