Single-Cell Transcriptomic Profiling of the O -Methyltransferase Characteristics of Flavonoid Biosynthesis in Ziziphus jujuba var. spinosa Fruit

Abstract Sour jujube (Ziziphus jujuba var. spinosa) is a valuable medicinal and edible fruit owing to its health-promoting properties. Methoxylated flavonoids enhance their value by improving stability and bioavailability. However, the cellular basis of their biosynthesis remains unclear. We constructed a single-nucleus transcriptomic atlas of sour jujube fruit. Parenchyma cells were identified as the primary sites for flavonoid biosynthesis and their O-methylation, characterized by high expression of phenylpropanoid pathway genes and O-methyltransferases (OMTs). Among the 48 identified OMTs, four (ZjOMT1–4) exhibited high expression, phylogenetically clustered with known functional OMTs, and localized to the cytoplasm; they were further characterized by enzyme activity assays. Notably, ZjOMT1 demonstrates moderate substrate promiscuity, catalyzing the methylation of various substrates, including quercetin, quercitrin, and rutin. Our findings reveal the cellular architecture of flavonoid metabolism in sour jujube fruit, providing genetic resources for breeding varieties with enhanced flavonoid profiles to develop functional foods and nutraceuticals.