Citrus reticulata ‘Chachiensis’ contributes its fruit peel to the raw material of ‘Guangchenpi’, is renowned for its distinctive medicinal and aromatic properties, and has been utilized for hundreds of years. However, the molecular and metabolic mechanism underlining the properties remains unknown. In this study, dimethyl anthranilate was uniquely detected in ‘Chachiensis’ fruit peel compared to other mandarin cultivars and was further validated as the characteristic metabolic biomarker based on orthogonal partial least squares discrimination analysis analysis. Two SAMTs genes, CreSAMT1 and CreSAMT2, were screened by combined volatile profiling and transcriptome sequencing. CreSAMT1 could catalyze the methylation of N-methyl-2-aminobenzoic acid to synthesize dimethyl anthranilate, and its constant expression contributes to the specific accumulation of dimethyl anthranilate in ‘Chachiensis’, which was activated by CreERF35 and CreZAT11. While CreSAMT2 is highly expressed in citrus flowers and is responsible for catalyzing anthranilate to form methyl anthranilate, the main floral volatiles. Moreover, the involvement of transcription factors such as ERF were speculated in regulating its volatiles biosynthesis. The study provides a theoretical basis to elucidate the volatile metabolism, and to improve the aromatic citrus industry.

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