Objectives:Terpenes are important volatile organic compounds that impact fruit aroma and flavor quality.Terpene synthases(TPSs)are the key enzymes responsible for the biosynthesis of basic backbone structure of terpenes.The identification and characterization of TPSs are critical for comprehending the biosynthesis of terpenes in fruit.Materials and Methods:The present study utilized cultivated peach(Prunus persica L.Batsch)as materials.RNA-sequencing was employed to investigate the expression profiles of PpTPSs during fruit ripening and in response to hormone and temperature treatments.Enzyme activities of PpTPSs were assessed using different substrates.Results:Here,we show that peach contains 38 TPS genes,with 24 members in the TPS-a cluster.Transcriptome analysis revealed that the expression of PpTPSs in peach fruits was regulated by environmental factors such as UV-B light and low temperature,as well as by phytohormones such as ethylene and methyl jasmonate.After analyzing the expression of 38 PpTPSs in peach fruit developmental stages and different tissues,we screened and cloned six new highly expressed TPS genes.Subcellular localization showed that PpTPS13 and PpTPS23 were localized in the plastid,whereas PpTPS12,PpTPS22,PpTPS25,and PpTPS28 were localized in the cytoplasm.Heterologous expression of PpTPSs in Escherichia coli followed by the enzymatic assays revealed that only fourTPSs(PpTPS12,PpTPS22,PpTPS25,and PpTPS28)were active in vitro.Using GPP and FPP as substrates,these PpTPSs were able to synthesize an array of volatile terpenes,including 15 monoterpenes such as geraniol,camphene,pinene,borneol and phellandrene,and 14 sesquiterpenes such as farnesene,nerolidol and α-bergamotene.Conclusions:Our results identify target genes for engineering to increase the production of volatile terpenes and thereby improve fruit quality.
Background:Ophiopogon japonicus(L.f)Ker-Gawl.growing in Zhejiang is recognized as the Dao-di medicinal herb for the production of Ophiopogonis Radix.Borneol-7-O-[β-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside,a prominent pharmacologically active compound,serves as a marker distinguishing O.japonicus in Zhejiang from those in other geographical areas.It is synthesized from borneol through glycosylation,with terpene synthase(TPS)being the critical enzyme catalyzing the conversion of terpene precursors into borneol.Objective:The aim of the study was to define key genes involved in biosynthesis of borneol in O.japonicus.Methods:The candidate terpene synthase genes were identified from the root and leaf transcriptome data of O.japonicus in Zhejiang and the functions of these enzymes were identified using engineered Escherichia coli.Results:This study developed a rapid expression system for monoterpene and sesquiterpene synthases based on engineered E.coli.Seven terpene synthase genes(OjTPS1 to OjTPS7)were identified in different terpene synthase subfamilies,including 2 from TPS-a,4 from TPS-b,and 1 from TPS-g.Biochemical analysis using an engineered system E.coli demonstrated that all the 7 terpene synthases produced monoterpenes,and OjTPS3,OjTPS5,and OjTPS6 also yielded sesquiterpenes.Conclusions:These 7 terpene synthases produced 13 monoterpenes and 8 sesquiterpenes.Notably,OjTPS1 produced borneol establishing the groundwork for elucidating the biosynthetic pathways of borneol-7-O-[β-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside and other volatile oil components.
