Y. Shotland - Shamoon College of Engineering, Department of Chemical Engineering, Beer Sheva, Israel
J. M. Hage - University of Calgary, Department of Biological Sciences, Calgary, Canada
P. J. Facchini - University of Calgary, Department of Biological Sciences, Calgary, Canada
Khat (Catha edulis) is a perennial shrub chewed for their psycho-stimulating properties common in the Middle East and Eastern Africa.

Khat (Catha edulis) is a perennial shrub chewed for their psycho-stimulating properties common in the Middle East and Eastern Africa. Among the active compounds in khat are the phenylpropylamino alkaloids such as cathinone that accumulates in young leaves. In addition to the alkaloids produced, leaves also accumulate various classes of terpenoids including volatile mono- and sesquiterpenes as well as non-volatile terpenoid-derived catheduline alkaloids. Sesquiterpenes are accumulated in many plant species in response to mechanical injury or pathogen infection. In this work we characterized the volatile terpenoids present in khat leaves in response to harvesting and artificial wounding. Leaf volatiles were sampled using solid-phase microextraction (SPME) coupled to GC-MS. The main sesquiterpenes in intact leaves were (E)-caryophyllene, α-copaene, α-cubebene, α-humulene, α-muurolene and δ- cadinene. Wounding the leaves resulted increased total sesquiterpene content, especially in the levels of (E)-caryophyllene and α-copaene. To test the potential biosynthetic activity for sesquiterpene formation after wounding, crude protein extracts were incubated with farnesyldiphosphate as a substrate and the sesquiterpenes produced in vitro were identified using GC-MS. Increased biosynthetic potential for (E)-caryophyllene, elemol, α-eudesmol as well as, b-bisabolene and α-(Z)-bergamotene was detected in extracts from wounded leaves as compared to controls. To get a broader view of genes that are up or down regulated in wounded leaves, we performed deep RNA sequencing of samples from wounded and non-wounded khat leaves. The analysis resulted in the identification of several novel putative terpene synthase genes. Functional expression of three of these genes indicated that they encoded proteins that catalyze the production of α-copaene and α-elemene (CeTps17580), elemol, α-eudesmol (CeTps13580), α-copaene, α-cubebene, γ-cadinene and δ-cadinene (CeTps6019). The identification and characterization of these genes together with the study of their expression levels as a result of wounding will help us to better understand the changes in the chemical composition ofkhat leaves upon wounding and the involvement of terpene biosynthesis in this process.