Functional characterization of terpene synthases MhTPS3 and MhTPS6 involved in linalool synthesis in Malus halliana flowers

Malus halliana, a highly valued ornamental tree species, has garnered considerable attention due to its distinctive floral fragrance. Nevertheless, the precise molecular mechanisms underlying floral fragrance biosynthesis remain poorly understood. In this study, a total of 69 floral volatile organic compounds were identified in M. halliana flowers using HS-SPME-GC×GC-TOF MS, with linalool emerging as the dominant constituent. Transcriptome profiling of flowers at different developmental stages revealed differential expression of six MhTPS genes. Notably, qRT-PCR analysis confirmed that the expression patterns of MhTPS3 and MhTPS6 were correlated with linalool emission dynamics. Phylogenetic analysis showed that MhTPS3 clusters in the TPS-g subfamily and MhTPS6 in the TPS-b subfamily. Subcellular localization experiments indicated that MhTPS3 was localized in both the nucleus and cytoplasm, while MhTPS6 was localized to the chloroplast. In vitro heterologous expression assays demonstrated that both MhTPS3 and MhTPS6 could utilize geranyl pyrophosphate (GPP) as a substrate to produce linalool. Furthermore, transient overexpression of MhTPS3 and MhTPS6 genes in crabapple flowers significantly increased linalool production. These findings identify MhTPS3 and MhTPS6 as key regulators of linalool biosynthesis in crabapple. This study provides mechanistic insights into the fragrance production in M. halliana and lays a molecular foundation for future genetic improvement of floral aroma.