Discussion
The expansion of a large number of duplicated genes and chromosomal rearrangements, which may contribute to the increase of metabolite content, is frequently accompanied by polyploidy, which is regarded as a key factor in species divergence (Ren et al., 2018; Van de Peer, Mizrachi, & Marchal, 2017), Polyploidy events were prevalent in dicotyledons and contributed to the accumulation of metabolites in plants. The polyploidization event in Hippophae rhamnoides may have contributed to the increased accumulation of fatty acid synthesis, AsA, and aldonic acid in its fruits (L. Yu et al., 2022). The positive effect of gene amplification on metabolite accumulation was discovered in the report of Artemisia argyi genome, which demonstrated its adaptability in the face of environmental stress (Miao et al., 2022). Our study uncovered that S. rosmarinus underwent an independent polyploidization event approximately 8.8 million years ago. Through analyzing the replication events of genes in the biosynthetic pathways of monoterpenes and diterpenes, we observed traces of a second WGD, suggesting that WGD-2 contributed to the expansion of genes involved in terpene biosynthesis, resulting in the mass accumulation of terpenoids in rosemary leaves.
Carnosol, the major active phenolic diterpene in S. rosmarinus , is found in various Mediterranean plants such as S. officinalis ,Thymus mongolicus and Origanum vulgare . Rosemary extracts was a source of high antioxidant compounds (Petiwala & Johnson, 2015). The biosynthetic pathways of diterpenoids in S. rosmarinus andS. miltiorrhiza diverged at the step of catalyzing the formation of 11-hydroxy-ferruginol. In S. rosmarinus , SrCYP76AK6–8 introduces a carbonyl group at C-20 to produce carnosaldehyde, which is further converted to carnosic acid and its derivatives (Ignea et al., 2016; Scheler et al., 2016). In S. miltiorrhiza,SmCYP76AK1 encodes an enzyme with hydroxylation activity at C-20 that primarily produces 11,20-dihydroxy-ferruginol for further biosynthesis of tanshinone using 11-hydroxy-ferruginol as a substrate (Guo et al., 2016). CYP76AK1 and CYP76AK6–8 were key enzymes in the same protein subfamily controlling the biosynthesis of diterpenoids inS. rosmarinus and S. miltiorrhiza , repectively, as a result of the evolutionary divergence of the ancestral CYP76AKgene (Bathe et al., 2019). Intrerestingly, CYP76AK1 was not identified in S. rosmarinus , and CYP76AK6–8 were not identified in S. miltiorrhiza . The results of the genealogical evolution of the genus Salvia indicated that the Salvia in Europe differentiated earlier than Salvia in Eastern Asia (Hu et al., 2018). CYP76AK1 and CYP76AK2 evolved from CYP76AK6and CYP76AK8. The evolutionary direction of CYP76AK6 andCYP76AK8 to CYP76AK1 and CYP76AK2 was consistent with the genealogical evolution of Europe Salvia to Eastern AsiaSalvia , which laid the genetic basis for structural differences in diterpenes.