Analysis of the DXS Pan-Gene Family Reveals Evolutionary Conservation and Regulatory Divergence Underpinning Terpenoid Metabolism in Lauraceae

1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the rate-limiting step of the plastidial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway and governs carbon flux toward terpenoid biosynthesis. The Lauraceae constitute an economically important plant lineage characterized by specialized terpenoid metabolism and divergent essential-oil chemotypes; however, the evolutionary dynamics and functional divergence of the DXS gene family across this family remain poorly understood. Here, we performed a comprehensive pan-genomic survey of the DXS family across 14 Lauraceae genomes, integrating phylogenomics, synteny analysis, selection-pressure assessment, structural-variation profiling, spatiotemporal expression profiling, and heterologous functional validation. We identified 92 DXS genes assigned to six orthogroups. DXS2 and DXS4 were conserved ohnologs likely derived from the Lauraceae-specific whole-genome duplication, whereas DXS5 and DXS6 underwent recent lineage-specific duplications. All DXS orthogroups were under purifying selection (Ka/Ks < 1), with divergent selection intensities among subclades within Clade 2. Structural variations in DXS genes were predominantly located in introns and untranslated regions rather than coding regions. Spatiotemporal expression profiling in Camphora officinarum revealed distinct tissue-specific expression patterns: CoDXS2 was specifically expressed in developing fruits, CoDXS4 exhibited flower-bud-specific expression, and CoDXS6 was highly expressed in developing leaves and fruits. Notably, CoDXS6 transcript abundance showed a strong positive correlation with monoterpenoid accumulation (r = 0.945, p < 0.001), and functional assays confirmed its canonical catalytic activity. Furthermore, CoDXS6 exhibited 6.32- to 9.18-fold higher leaf expression in terpenoid-dominant chemotypes compared with phenylpropanoid-dominant chemotypes. Collectively, retention following recent whole-genome duplication, recent genome-specific duplications, and regulatory variations jointly drove the functional divergence of Lauraceae DXS genes, highlighting CoDXS6 as a key contributor to leaf monoterpenoid biosynthesis in C. officinarum.