<i>CitNOR-like1</i> is conserved in the regulation of ascorbic acid accumulation in plants

L-Ascorbic acid (AsA), commonly known as Vitamin C (Vc), is indispensable to humans and is crucial for maintaining redox homeostasis during plant growth and development, fruit ripening, and stress responses. Citrus species are diverse and widely distributed, and their fruits are vital dietary sources of Vc. However, the regulatory mechanism underlying AsA accumulation in citrus fruit remains unclear. In this study, we observed that Citrus species exhibit significantly higher AsA content and Pectin methylesterase (PME) expression than Citrus-related genera in mature fruit pulp, with AsA levels and PME expression declining during fruit ripening. Functional validation confirmed that CitPME positively promotes AsA biosynthesis in citrus. Notably, a miniature inverted-repeat transposable element (MITE) insertion in mandarin and sweet orange CitPME promoters contributed to higher promoter activity than that observed in Citrus-related genera. Furthermore, CitNOR-like1, a NAC transcription factor (TF) homolog of the tomato (Solanum lycopersicum) SlNOR-like1 involved in fruit ripening, was identified as a negative regulator of AsA accumulation. CitNOR-like1 expression increased during fruit ripening and resulted in repressed CitPME transcription, exhibiting a stronger repressive effect on PME promoters from Citrus-related genera than those from Citrus. Heterologous overexpression of CitNOR-like1 significantly decreased AsA content in tobacco (Nicotiana tabacum) and tomato and promoted fruit ripening in tomato. Collectively, this study identified a key TF involved in regulating AsA accumulation in citrus fruit. These findings shed light on the genetic regulation of AsA and offer a potential strategy to modulate fruit ripening without compromising fruit AsA content.