Molecular Mechanism Of UGT74AF3 Mediated Glucosylation Of 4-Hydroxy-2,5-dimethylfuran-3(2H)-one In Camellia Sinensis

Volatile compounds are always viewed as an important factor related to tea quality,which directly affect people’s consumption behavior.4-Hydroxy-2,5-dimethyfuran-3(2H)-one(HDMF)with a strong caramel-like aroma widely exist in strawberry,grape and other fruits and is considered as an important aroma compound.Besides,HDMF is also regarded as an important aroma compound which contributes to the caramel-like note of some teas.For a long time,HDMF has been always considered as the product of Maillard reaction.However,its biosynthesis and metabolism in tea plants are still unknown.Here,HDMF glucoside was identified as a native metabolite in the tea plant.And then,a novel glycosyltransferases(UGTs)gene,named UGT74AF3,was selected from a lot of UGTs genes of the tea genome according to the features of subfamily of UGTs.UGT74AF3 a and UGT74AF3 b proteins can specifically catalyze the glucoside formation of HDMF.Afterwards,in vivo and in vitro experiments were carried out in this study.These results pave the way for the elucidation of molecular mechanism of HDMF synthesis.The main work and results are summarized as follows:1)The leaves of tea plants(shuchazao)were used as materials to study whether HDMF glucoside was present in tea plants.HDMF glucoside was identified as a native metabolite by feeding aroma compound and LC-MS in tea plants.2)UGT74AF3a/b proteins expression and purification were carried out in this study.Afterwards,activity screening of UGT74AF3a/b proteins with different substrates was carried out.The results showed that UGT74AF3a/b proteins can specifically catalyze the glucoside formation of HDMF.3)Sequence comparison between UGT74AF3 a and UGT74AF3 b was carried out in this study.And then,a single A456 V mutation of UGT74AF3 b was carried out.Afterwards,the enzymatic activity assay of UGT74AF3 and mutant proteins was carried out.The results showed that the residue A456 V played a key role in the sugar-donor preference and enzyme activity of UGT74AF3.4)Transient overexpression of UGT74AF3 a in tobacco was carried out in this study.The result showed that UGT74AF3 a can catalyze the glucoside formation of HDMF.5)UGT74AF3a suppression was carried out in a tea cultivar.Then,the concentration of HDMF glucoside and the transcript levels of UGT74AF3 a were detected in different tea cultivars.The results showed that UGT74AF3 a can not only catalyze the glucoside formation of HDMF in a tea cultivar,but also has the same function in different tea cultivars.