Lineage-specific Emergence and Multi-layered Regulation of Limonoid Biosynthesis in Citrus

Limonoids are triterpenoid phytochemicals that contribute to delayed bitterness in citrus fruit, enhance pest resistance, and exhibit a wide range of bioactivities with nutraceutical and agrochemical potential. Despite their importance, the genetic and regulatory basis underlying limonoid biosynthesis in citrus remains poorly understood. Here, by integrating targeted metabolite profiling across 134 citrus and related accessions with comparative genomic analyses of 12 representative species from the orange subfamily (Aurantioideae), we show that the emergence of limonin, nomilin, and obacunone correlates with the citrus-specific acquisition of three downstream biosynthetic genes, CYP88A51, CYP716AC1, and limonoid furan synthase (LFS), while the upstream oxidosqualene cyclase 1 (OSC1) remains conserved across lineages. We further identify CsbHLH92 as a key transcriptional activator that directly regulates these genes, and its overexpression led to dramatic increases in limonin and nomilin levels. Moreover, CsbHLH92 activity is attenuated by CsMPK7-mediated phosphorylation, contributing to the downregulation of limonoid biosynthesis during leaf senescence, when senescing leaves are generally less preferred by insect pests. Together, our findings uncover how lineage-specific gene emergence and multi-layered regulation collectively shape the specialized metabolism of limonoids in citrus. These insights provide metabolic engineering targets for improving citrus flavor, enhancing pest resistance, and boosting the production of high-value bioactive compounds.