The AP2 transcription factor CsAIL6 negatively regulates citric acid accumulation in citrus fruits by interacting with a WD40 protein CsAN11

Citric acid accumulation is an essential process in citrus fruits that determines fruit flavor and marketability. The MBW complex transcription factor genes, CsAN11, CsAN1, and CsPH4 play key roles in regulating citric acid accumulation. Although how to regulate CsAN1 or CsPH4 was widely investigated, studies on the regulation of CsAN11 are scarce. In this study, we characterized the AP2/ERF (APETALA2/ethylene response factor) transcription factor gene CsAIL6, which is lowly expressed in high-acid citrus varieties and highly expressed in low-acid citrus varieties. Overexpressing CsAIL6 obviously decreased the citric acid content in citrus fruits, calli or tomatoes, whereas silencing CsAIL6 significantly increased the fruit citric acid content. Additionally, transcript levels of CsAN11, CsAN1, and CsPH4 were significantly increased by silencing CsAIL6; only the CsAN11 transcript level was significantly decreased by overexpressing CsAIL6. Similarly, only the tomato AN11 (SIAN11) transcript level in CsAIL6 stably overexpressing fruits was markedly lower than that in WT fruits. Further experiments revealed that overexpressing CsAN11 significantly increased the organic acid content but had no obvious influence on the CsAIL6 transcript level; in addition, CsAIL6 only interacts with CsAN11, rather than with CsAN1 and CsPH4 of the MBW complex. Taken together, our findings verified that CsAIL6 negatively regulates citric acid accumulation through directly interacting with the WD40 protein CsAN11, which provides a new mechanism for citric acid accumulation in fruits through the regulation of the MBW complex.