Cloning and functional characterization of the terpene synthase genes CiaTPS1 and CiaTPS2 from Chrysanthemum indicum var. aromaticum Q. H. Liu et S. F. Zhang

Chrysanthemum indicum var. aromaticum, an aromatic plant endemic to the Shennongjia region, is characterized by its distinct whole-plant fragrance, primarily derived from terpenoid compounds. Terpene synthases (TPSs) catalyze the formation of diverse terpene skeletons and play a central role in volatile terpenoid biosynthesis. Based on genomic data from C. indicum var. aromaticum, two TPS genes, CiaTPS1 and CiaTPS2, were cloned and subjected to bioinformatic characterization, subcellular localization, and functional enzymatic assays. Results showed that the open reading frame (ORF) of CiaTPS1 was 1 722 bp in length, encoding a 573-amino-acid protein, while the ORF of CiaTPS2 spanned 1 725 bp, encoding a 574-amino-acid protein. Both CiaTPS1 and CiaTPS2 contained conserved domains and signature motifs characteristic of plant TPSs and were classified within the TPS-a subfamily. Subcellular localization analysis revealed the dual presence of CiaTPS1 and CiaTPS2 proteins in the cytoplasm and nucleus. Recombinant CiaTPS1 and CiaTPS2 were produced via heterologous expression in Escherichia coli and subsequent purification. Both enzymes catalyzed the conversion of geranyl diphosphate (GPP) into eight distinct monoterpenes, with α-terpinene as the predominant product. CiaTPS1 additionally catalyzed the conversion of farnesyl diphosphate (FPP) into 10 sesquiterpenes, with 1-methyl-4-(6-methylhept-5-en-2-yl)cyclohexa-1,3-diene as the major compound, whereas CiaTPS2 generated four FPP-derived sesquiterpenes, with zingiberene as the primary product. These findings provide a molecular basis for understanding the biosynthesis of volatile terpenoids in C. indicum var. aromaticum and offer a foundation for future metabolic and evolutionary studies.