Guangdong Citri Reticulatae Pericarpium from the dry and mature peel of Citrus reticulata ‘Chachi’ (CRC) is a well-known medicinal and food material in Asia. The main propagation methods of CRC are layerage and grafting. It is generally considered that the quality of CRC from layerage is superior to that obtained from plants propagated by grafting. Nevertheless, the effects of layerage and grafting on the biosynthesis of flavonoid (main bioactive ingredients) in the peel of CRC remain unknown. Here, metabolomic analyses revealed the effects of layerage, self-grafting, and heterografting (Citrus limonia as rootstock) on flavonoid biosynthesis in CRC from two main harvesting periods, CRCV (Citri Reticulatae Chachiensis Viride) and CRCR (Citri Reticulatae Chachiensis Reddish). Compared with CRCR, CRCV exhibited a higher content of flavonoids. Grafting CRC onto C. limonia exhibited a higher content of hesperidin, nobiletin, tangeretin, narirutin, demethylnobiletin, and sinensetin than layerage and self-grafting. This increase can be attributed to the upregulation of genes involved in flavonoid synthesis. Further, the transcription factor CrcMYBF1 was identified within the gene coexpression network and is confirmed to be significantly induced by methyl jasmonate (MeJA) and upregulate the expression of Crc1,6RhaT through interacting with its promoter region, thereby boosting the biosynthesis and accumulation of hesperidin. In summary, our findings provide mechanistic insights into the coordinated regulation of hesperidin biosynthesis via MeJA-inducing CrcMYBF1 in CRC. Our study is expected to provide a theoretical basis for CRC propagation and cultivation.

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