Monoterpene synthases contribute to the volatile production in tana (Zanthoxylum ailanthoides) through indigenous cultivation practices.

Tana (Zanthoxylum ailanthoides), a perennial deciduous species in the Rutaceae family, possesses leaves with a unique fragrance that indigenous peoples incorporate into their traditional cuisine. In Kalibuan, the cultivated tana trees were pruned repeatedly to maintain a shorter height, which led to the growth of new leaves that were spicier and pricklier. Tana leaves contain a range of volatile terpenoids, and the pungent aroma may arise from the presence of monoterpenoids. To gain insight into the biosynthetic pathway, five candidate monoterpene synthase genes were cloned and characterized using a purified recombinant protein assay. The main product of Za_mTPS1, Za_mTPS2, and Za_mTPS5 is sabinene, geraniol, and (E)-ß-ocimene, respectively. The main product of Za_mTPS3 and Za_mTPS4 is linalool. Real-time PCR analysis revealed that Za_mTPS1 and Za_mTPS5 are expressed at higher levels in prickly leaves of cultivated tana, suggesting that they may contribute to the distinctive aroma of this plant.

Sesquiterpene Synthases of Zanthoxylum ailanthoides: Sources of Unique Aromas of a Folklore Plant in Taiwan.

Zanthoxylum ailanthoides is a traditional spice crop in Taiwan with unique smells and tastes that differ between prickly (young) and nonprickly (mature) leaves. Different volatile terpenes between prickly young and nonprickly mature leaves were identified and considered to be one of the sources of their aromas. A transcriptome database was established to explore the biosynthesis of these compounds, and candidate terpene synthase genes were identified. The functions of these synthases were investigated using recombinant protein reactions in both purification and coexpression assays. ZaTPS1, ZaTPS2, and ZaTPS3 are germacrene D synthases, with different amino acid sequences. The main products of ZaTPS4 are trans-α-bergamotene and (E)-ß-farnesene, whereas ZaTPS5 forms multiple products, and ZaTPS6 produces ß-caryophyllene. ZaTPS7 forms monoterpene (E)-ß-ocimene and sesquiterpene (E,E)-α-farnesene. Reverse transcription PCR of ZaTPS gene expression in young and mature leaves revealed that ZaTPS1 was responsible for the mellow aroma in mature leaves. The expression of ZaTPS6 suggested that it plays a role in the background aromas of both types of leaves. Our findings deepened the understanding of the volatile compounds of Z. ailanthoides and revealed the source of its unique aromas by clarifying the biosynthesis of these compounds.