Functional Characterization of AmGPPS/GGPPS Gene Family in Antirrhinum majus and the Regulatory Role of AmGPPS6 in Floral Scent Variation

Geranyl diphosphate synthase (GPPS) is a key enzyme in the plant isoprenoid metabolic pathway and regulates the biosynthesis of volatile monoterpenes. It plays an important role in the biosynthesis of floral volatile terpenoids (FVTs) and inter-cultivar variation in snapdragon. Despite its importance in floral scent formation, the GPPS/GGPPS gene family in snapdragon (Antirrhinum majus L.) has not been systematically characterized. In this study, nine GPPS/GGPPS family members were identified at genome-wide level. These include six AmGPPS and three AmGGPPS genes. Phylogenetic analysis grouped them into distinct subfamilies. We further analyzed their chromosomal locations, gene structures, conserved protein motifs, and promoter cis-acting elements. These results revealed both conservation and functional divergence within the gene family. To explore their functional roles, we compared gene expression profiles at the full flowering stage. This comparison was performed between strongly scented cultivar (Am3) and the weakly scented cultivar (Am5). Among all candidates, AmGPPS6 showed the most significant differential expression. Further, functional validation was conducted using transient overexpression and virus-induced gene silencing (VIGS). Overexpression of AmGPPS6 significantly increased terpenoid production. Total floral volatile terpenoids (FVTs) increased by 1.4 fold. Both monoterpene and sesquiterpene emissions were enhanced. In contrast, silencing of AmGPPS6 markedly reduced the emission of key monoterpenes such as ocimene and its isomers. Sequence analysis showed that AmGPPS6 shares 67.04% identity with canonical GPPS small subunit (GPPS.SSU). However, it lacks the conserved catalytic DDx2-D motif. This suggests that AmGGPPS2 is not catalytically active. Instead, it likely functions through heterodimer with AmGGPPS2. This interaction is supported by coordinated transcriptional expression patterns. Additionally, natural sequence polymorphisms were identified in GPPS.SSU. These variations, rather than those in GPPS.LSU, appear to drive differences in monoterpense emission between cultivars. In conclusion, AmGPPS6 in a key regulator of floral scent biosynthesis in snapdragon. This study provides new insights into functional roles of GPPS/GGPPS genes. It also offers valuable gene targets for the molecular breeding of aromatic traits in ornamental plants.