Bioinformatics and Biochemical Analysis of Wound-Induced Sesquiterpene Biosynthesis in Khat (Catha edulis)

Yoram shotland, Chemical Engineering, SCE - Shamoon College of Engineering, Beer Sheva, Israel
Einat Bar, Department Of Vegetable Crops, Newe Yaar Research Center, Agricultural Research Organization, The Volcani Center, Ramat Yishi, Israel
Rachel Davidovich-Rikanati, Department Of Vegetable Crops, Newe Yaar Research Center, Agricultural Research Organization, The Volcani Center, Ramat Yishi, Israel
Vered Shain, Chemical Engineering, Sce - Shamoon College Of Engineering, Beer Sheva, Israel
Jillian M Hagel, Department Of Biological Sciences, University Of Calgary, Calgary, Calgary, Canada
Peter J. Facchini, Department Of Biological Sciences, University Of Calgary, Calgary, Calgary, Canada
Natalia Dudareva, Department Of Biochemistry, Purdue University, West Lafayette, Usa
Efraim Lewinsohn, Department Of Vegetable Crops, Newe Yaar Research Center, Agricultural Research Organization, The Volcani Center, Ramat Yishi, Israel

Khat (Catha edulis) leaves are commonly chewed for their mild psycho-stimulating properties, in parts of the Middle East and Eastern Africa. The psychoactive compounds in khat are thought to be the cathamine alkaloids such as cathine and cathinone but leaves also accumulate mono- and sesquiterpenoids and terpenoid-derived catheduline alkaloids.

Sesquiterpenes are accumulated in many plant species in response to mechanical injury or pathogen attacks. Metabolic profiling of volatile terpenoids present in khat leaves indicated that the main sesquiterpenes accumulated in intact leaves were (E)-caryophyllene, alpha-copaene, alpha-cubebene, alpha-humulene, alpha-muurolene and delta-cadinene. Wounding the leaves resulted in increased total sesquiterpene content, and increases in the levels of (E)-caryophyllene and alpha-copaene. Crude protein extracts incubated with farnesyl diphosphate as a substrate indicated an increased biosynthetic potential for (E)-caryophyllene, elemol, alpha-eudesmol beta-bisabolene and alpha-(Z)-bergamotene in extracts from wounded leaves as compared to non-wounded leaves. 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 formation of various sesquiterpenes from farnesyl diphosphate in vitro. Our results will help us to better understand the wound responses in khat leaves and the involvement of terpene metabolism in this process.