CgLS mediates limonene synthesis of main essential oil component in secretory cavity cells of Citrus grandis ‘Tomentosa’ fruits

d-Limonene is the predominant component of essential oil from Exocarpium Citri Grandis, known for its antibacterial, antioxidant, insecticidal, and anti-inflammatory properties. The synthesis, transport, and accumulation of d-limonene in Citrus grandis 'Tomentosa' fruits are regulated by limonene synthetase (LS) and its associated regulatory genes. This study addresses the gap in understanding the spatiotemporal cytological changes of LS and its regulatory genes. Through cytochemical techniques, we investigated the distribution of essential oil in the plastids, endoplasmic reticulum, and vacuoles of secretory cavity cells. We identified the LS-encoding gene CgLS via transcriptomics and demonstrated in vitro that CgLS catalyzes the formation of d-limonene from geranyl diphosphate (GPP). Transient overexpression of CgLS increased monoterpene limonene accumulation, while TRV virus-induced gene silencing reduced it. CgLS expression levels and d-limonene content showed spatiotemporal consistency with fruit development, with in situ hybridization revealing predominant expression in secretory cavity cells. Immunocytochemical localization indicated that CgLS is primarily located in the endoplasmic reticulum, plastids, and vacuoles. Our findings suggest that CgLS is translated in the endoplasmic reticulum and transported to plastids and vacuoles where d-limonene synthesis occurs. This study provides comprehensive insights into the characteristics of CgLS and its role in d-limonene synthesis at the tissue, cellular, and molecular levels in C. grandis 'Tomentosa'.