Studing On The IBMP Biosynthesis And Intermediate Metabolites In Vitis Vinifera

3-Alkyl-2-methoxypyrazines（MPs）is one of the main sourece of herbaceous and vegetal sensory characteristics compounds which contribute to the aroma and flavor of many grapes varities and wines.The biosynthetic pathway of MPs has not been fully elucidated,and only a few studies have focused on it.To date,there are two proposed pathways of MPs biosynthesis,one suggested that the initial step involve amidation of a amino acid（hereinafter referred to as “Amino acid amidation pathway”）,the other believed that the initial step in pyrazine biosynthesis is the condensation of two amino acids to a cyclic dipeptide（hereinafter referred to as “Amino acid condensation pathway”）.Based on the two hypothesis of MPs biosynthesis,we explored the key intermediate of IBMP biosynthesis and the origin of IBMP in grape berries.The proposed precursor with 13 C labled(2-¹³C-L-Leu)was incorporated into preverasion clusters and leaves of Cabernet Sauvignon in situ,respectively;The proposed precursor（L-Leu）and key intermediates（2-Amino-4-methylpentanamide,AMPA;2,5-dicarbonyl-3-isobutyl-piperazine,DCIP）of IBMP biosynthesis were incorporated into preverasion clusters,respectively.IBMP concentration and the main Vv OMTs gene expression levels were monitored.Besides,we incorporated 2-¹³C-IBMP into the preverasion and postverasion grapes of Cabernet Sauvignon in vitro,respectively.The degradation pathway of IBMP and the effect of temperature and light on IBMP degration were explored.The main findings are as follows:（1）Using the 2-¹³C-L-Leu as the only tracer to labled the Cabernet Sauvignoncluster in situ,it was found that the IBHP in grape berry skin was also labled with 13 C,which further confirmed that IBHP was indeed the key intermediate of IBMP biosynthesis.Especially it confirmed that L-Leu was the precursor of IBMP biosynthesis in grape berry.The 13 C atom% of AMPA was slightly higher than natural abundance and always lower than that of IBHP during the whole treatment period suggest that,to some extent,the “Amino acid amidation pathway” was one of the pathway of IBMP biosynthesis in grape.（）（2）The effect of L-Leu and AMPA incorporation into the Cabernet Sauvignon grape in preverasion in situ was ascertained.The results showed that the IBMP concentration and the expression levels of Vv OMT1 and Vv OMT3 were positively related to L-Leu and AMPA incorporation after verasion,and the increased of IBMP content in AMPA treatment was earlier than that of in L-Leu treatment,this result was further confimed that the “Amino acid amidation pathway” of IBMP biosynthesis may exist in grape berry.（3）The compound 2,5-dicarbonyl-3-isobutyl-piperazine（DCIP）was detected in berry skin and showed a trend of continuous decline during the grape maturity,which similar with the IBMP.Besides,incorporated the DCIP into the Carbernet Sauvignongrape in situ,it was found that the IBMP concentration and Vv OMT3 expression level was significant increased in matured grapes,the results showed that DCIP may also involved in the biosynthesis of IBMP in grape berry.（4）Incorporated the 2-¹³C-L-Leu into the Cabernet Sauvignon leaves in situ,the high abundance of 13 C labeled L-Leu and IBHP,and slightly 13 C labeled AMPA in corresponding grape berry skin were detected.The result showed that the IBMP of grape was biosynthesis in situ,and also transported precursor and intermediate from leaves.（5）Using the 2-¹³C-IBMP as the only tracer to labeled the Cabernet Sauvignon grapes in vitro,it was found that the IBHP in berry skin was labeled with high abundance of 13 C,which further confirmed that IBHP was indeed the key intermediate of IBMP degradation.Furthermore,another compound,2,5-dicarbonyl-3-isobutyl-piperazine（DCIP）was also detected with high abundance of 13 C in berry skin at the same time in our experiment,which showed that DCIP was also derived from the IBMP degradation in grape.These results provided important information to explore the degradation pathway of IBMP in grape berry.（6）The high abundance of 13 C labeled IBHP and DCIP were also founded in 2-¹³C-IBMP labeled preverasion grape berries which was in a biosynthesis phase,these results showed that the IBMP metabolic in grape berry before verasion may not simply be a biosynthesis process,but also coexist with degradation;The IBMP content in grape after L-Leu and AMPA treated was significantly increased in postverasion until maturity,and DCIP treated was significantly increased in maturity which were in a degradation phase.These results showed that IBMP metabolism after verasion may not simply be a degradation process,but may also coexist with biosynthesis.（7）Light and temperature were both control the IBMP and IBHP degradation in grape berry,and the effect of temperature was more prominent.