| Terpenoids are the largest and most diverse class of plant natural products,which are synthesized by terpene synthases(TPSs).Monoterpenoids and sesquiterpenoids are volatiles playing important roles in fruit aroma quality.For citrus fruit,terpenoids are the main volatile components.Difference in volatile composition between citrus species is the main cause of their characteristic aroma.Citrus medica is one of the progenitor species in citrus with biological and commercial importance.Finger citron(C.medica L.var.sarcodactylis Hort)belongs to C.medica,and is accepted by consumers due to its unique fragrance.Essential oils extracted from finger citron had been successfully applied in industry.However,the characteristic volatiles of finger citron different from other citrus fruits still remain unclear.Moreover,spatial and temporal distribution of volatile terpenoids in developing flowers,leaves and fruits need to be investigated.Identification of volatile formation related genes and transcriptional regulatory networks in finger citron are rarely understood.In this study,finger citron was used as material,comprehensive study was applied to explore terpenoid metabolism using metabolomic,transcriptomic and molecular techniques.The main results are as follows:1.A total of 62 volatiles were identifed from finger citron tissues using headspace solid phase microextraction(HS-SPME)combined with gas chromatography-mass spectrometry(GC-MS)techniques.The most abundant compound is limonene,representing 41.79%and 58.36%of mature leave and fruit,respectively.The total content of volatiles increased with the maturation of organs.Mature fruit has the highest volatile content(26.25 mg/g),which is about 4 times higher than that of flower bud.Venn diagram showed that 33 volatiles were detected in all three organs.Using the partial least squares(PLS)discriminant analysis,24 compounds with a VIP higher than 1.0 were identified,which play important roles in separating finger citron organs.Among these volatiles,six compounds could be used as potential markers for separating citron and other Citrus species,which was in agreement with citrus taxonomy.These six compounds could be the characteristic volatiles of finger citron fruit.2.TPS genes and transcription factors associated with terpenoid formation were screened using a weighted co-expression network analysis analysis(WGCNA).A total of 32579 genes were detected by RNA-seq based on C.medica genome.There were 58 TPS gene family members,belonging to subfamily a,b,e/f and g.According to their transcript levels,16 TPS genes were mainly expressed in flower,25 TPS genes were mainly expressed in leave,and 17 TPS genes had the highest transcript levels in fruit.Transcription factors such as NAC,MYB,WRKY,bHLH,AP2/ERF and bZIP families were identified,whose expression patterns in finger citron organs during development were explored.WGCNA showed that genes involved in terpenoid biosynthesis were clustered in Module Red and Module Green,where three TPS genes and five TFs were screened as biologically important genes for volatile terpenoid formation in finger citron.3.Finger citron CmTPS1 is associated with the formation of sesquiterpene bicyclogermacrene.In the present study,CmTPS1 comprised of 1668 base pairs was cloned,belonging to TPS-a subfamily.Transiently transform experiment in tobacco leaf cells showed that CmTPS1 protein localized in the cytoplast.Accumulated concentration of bicyclogermacrene positively correlated with CmTPS1 transcripts in developing fruit,flowers and leaves of finger citron.The recombinant protein of CmTPS1 can utilize fanesyl pyrophosphate(FPP)as substrate to catalyze the formation of sesquiterpenes at 30? with the main product bicyclogermacrene.No volatile compounds were detected using geranyl diphosphate(GPP)as substrate.Transient overexpression analysis in finger citron leaf showed that increased expression of Cm TPS1 induced bicyclogermacrene accumulation. |
PDF Full Text Request