The characteristic bitter substances in ‘Shitougan’(Citrus reticulata Blanco cv. Manau Gan) fruit and the key genes regulating their synthesis

‘Shitougan’ is a distinctive germplasm resource of citrus found in the northern margin of the subtropical zone in southern Shaanxi, China. It differs from other citrus varieties in terms of appearance, taste, and aroma. The fruit exhibits a moderately sweet and sour flavor with a slightly bitter aftertaste. To elucidate the material basis and gene regulatory mechanisms underlying its unique bitterness, this study employed a combination of transcriptomics and metabolomics to dynamically analyze bitter metabolites and gene expressions associated with biosynthesis during three stages of ‘Shitougan’ fruit development. A total of 54 bitter compounds were identified from 952 metabolites, with flavanone compounds being the primary contributors to bitterness. Correlation analysis and partial least squares discriminant analysis revealed that neohesperidin and naringin serve as marker metabolites for its bitterness. Transient overexpression of five candidate genes ( CitPAL1 , CitPAL2 , CitC4H , CitCHI , and CitF3'H ) in both ‘Shitougan’ peel and tobacco leaves, along with genetic transformation analysis in tobacco, demonstrated that CitPAL2 , CitC4H , and CitCHI , which are upstream regulators in the flavanone synthesis pathway, first control the synthesis of naringenin and rapidly channel it into downstream metabolic pathways. This not only promotes the synthesis and accumulation of naringin and neohesperidin but also enhances the accumulation of rutin and procyanidin B2. These findings provide a theoretical foundation for the development of functional nutrients in ‘Shitougan’ fruit and the investigation of the biosynthetic mechanisms of bitter substances. • Non-targeted metabolomics identified bitter-tasting substances. • Neohesperidin and naringin identified as bitter taste markers in 'Shitougan' fruit. • CitPAL2, CitC4H, and CitCHI regulate the accumulationof naringin and neohesperidin in 'Shitougan' .