Influence Of Tea Plant Vesicle Transport And Glycosylation On Terpene Glycosides Accumulation

In plant,volatile terpenoids usually store in cells in the form of water-soluble glycosides.Until now,many glycosidically bound volatiles have been found in tea leaves,especially monoterpene glycosides,like geranyl and linalyl glycosides,which almost are highest abundant in tea leaves.Biosynthesis and accumulation of these glycosides in cells play a very important role on the aroma quality of black tea and oolong tea,and also have important biological function.Verify how these volatiles and their glycosides cross membranes and where they store not only can help to improve tea aroma quality,but also help to understand ways of volatilization and accumulation of volatiles.Vesicle transport is an important pathway for macromolecular compounds exchange intracellular and intercellular in plant,and research showed that inhibition of exocytosis could significantly increase the accumulation of terpene glycosides in plant.To study the probable storage site and transport pathways for volatile terpene glycosides,binary expression vectors of geraniol synthase gene ObGES,glycosyl transferase gene CsGT1,CsGT2,Qb-SNARE gene AtVTI12 and Cs VTI12,and RNA interference of R-SNARE gene NtVAMP72(MtVAMP721e-RNAi)were constructed.Tansient expressed one or several of these vectors in Nicotiana benthamiana leaves,and used SPME-GC-MS and UPLC-Q-TOFMS/MS to detect the amount of released geraniol and content of geranyl glycoside.At the same time,ObGES stably expressed tobacco was constructed and was carried out for research.Results show that high abundance of geranyl diglycoside were detected in ObGES transient expressed leaves,which indicates this gene has normal function.Compared with only expressed ObGES into tobacco leaves,the abundance of geranyl diglycoside significantly increased in ObGES and MtVAMP721e-RNAi co-expressed leaves,which indicates vesicle transport probably involved in volatilization of geraniol and the storage site of terpene glycoside may not be ectoplasm.In ObGES co-expressed with CsGTl and CsGT2 and ObGES co-expressed with CsVTI12 or AtVTI12 leaves,no obvious increment of the content of geranyl diglycoside was found,which might indicate Nicotiana benthamiana itself can totally glycosylates geraniol and transport them to vacuoles.In ObGES overexpressed Nicotiana tabacum,transcript levels of NtVTI12 and NtVAMP72c were significantly higher than in wild type,which further supporting that vesicle transport probably involved in volatilization of geraniol and the storage site of terpene glycoside may be vacuoles.Further more this research also found that wild type Nicotiana benthamiana leaves have geranyl and linalool diglycosides.But without adding β-glucosidase,geraniol and linalool were undetectable in wild type sample in GC-MS data,which indicates that this kind of β-glycosidases were in a very low level or low activity in Nicotiana benthamiana leaves.In blank control leaves which injected buffer,the abundance of geranyl diglycoside increased but linalyl diglycoside decreased,and geranyl glucoside was detected,which may caused by change of synthesis and/or activity of some glycosyl transferase and glycosidase after mechanical damage.Taken together,this study preliminary ascertain the transport pathway of volatile terpene and the storage site of its glycoside through regulating glycosyl transferase and vesicle fusion with plasma membrane or tonoplast.It provides theoretical basis and thoughts for future research of synthesis and transportation of glycosides.