The Roles Of Loxs In Synthesis Of Fruit Aroma Volatiles In Oriental Melon (Cucumis Melo Var.Makuwa Makino)

The oriental melon(Cucumis melo var.makuwa Makino)is very popular in customers because its outstanding aroma characteristic and nutrition.Among fruit qualities,flavor such as taste and aroma play key role in determination of consumers’ choice.Nowadays,research on melon aroma is more and more important.Aroma compounds in fruit mainly produce via amino and lipoxygenase pathway.In this study,we elucidate that there is a positive relationship between LOXs activity and aroma compounds production in oriental melon,and CmLOXs genes are involved in fruit aroma generation in melon.To this end,we tested four aroma types of melon that feature a thin pericarp:two aromatic cultivars of the oriental melons,’Yu Meiren’(YMR)and ’Cui Bao’(CB);a non-aromatic oriental pickling melon(C.melo var.conomon),’Shao Gua’(SHAO);and a non-aromatic snake melon(C.melo L.var.flexuosus Naud),’Cai Gua’(CAI).In the present research,function of LOX in aroma compounds production in oriental melon and effects of exogenous MeJA on LOX activity and expression of LOX gene family members during melon storage were investigated.Two LOX gene,CmLOX03 and CmLOX04 were cloned.Transient expression was applied to study function of CmLOX03 and CmLOX04 in straight chain esters production in oriental melon.The main results are described as followings:1.Four different types of melon showed different exhibited different morphological and physical characteristics during maturation.Approximately 87 volatile compounds,including esters,aldehydes,alcohols and acids,were detected in these four types of melons.The total aroma compounds content and ester content in YMR were the highest among the different types of melon.In order to visualize the differences among the four types of melon,principal component analysis was conducted to analyze these 87 compounds.SHAO and CAI separated from YMR and CB across PCI.Ethyl acetate,benzyl acetate,(E,Z)-2,6-nonadienal and menthol were four principal volatile compounds that affected the flavor of these four types of melon.Total LOX activity was highest in YMR,almost twice that of SHAO and CAI.This result is consistent with the content of total aroma aromatic compounds in different types of melon.These results suggest that LOX activity and ester production are positively correlated in melon.2.The expression patterns of all 18 CmLOXs genes in mature melon fruit differed by aromatic type.In our current investigations,we observed that CmLOX05,CmLOX10,CmLOX11 and CmLOX16 were highly expressed in YMR,thereby suggesting that these genes might be associated with higher LOX activity in highly aromatic melons.CmLOX08,CmLOX09 and CmLOX17 highly expressed in CB.The expression levels of CmLOX02,CmLOX03,CmLOX04,CmLOX12,CmLOX15,and CmLOX18 were highest in SHAO,thus indicating that these 6 genes may be important for the synthesis of alcohols via the LOX pathway in less aromatic melons.CmLOX07,CmLOX13,and CmLOX14 may be important for the synthesis of aldehydes in CAI.3.In our fruit-disk experiments,LA and LeA treatment significantly increased the accumulation of total aldehydes.More varieties and higher concentrations of volatile esters were detected upon LOX substrate treatment.The production of straight-chain esters such as ethyl acetate increased in response to LA and LeA treatments and decreased in response to inhibitor treatments.Our findings suggest that LOX activity positively correlates with the straight-chain ester contents in oriental melon.LOX pathway may be involved in ester formation in melons.CmLOX03,CmLOX04,CmLOX05,CmLOX11,CmLOX12,CmLOX15,CmLOX16 and CmLOX18 were up-regulated by the addition of two LOX substances,and they were down-regulated to different extents by the addition of LOX inhibitors.This pattern suggests that LOX substrate accumulation affects CmLOXs gene expression in melons and that CmLOXs gene expression may positively correlate with straight-chain ester production.In addition,the highest expression level of CmLOX04,CmLOXll,CmLOX15 and CmLOX05,was observed at 5 DAP and root,which indicates that they may play a limited role in ester production.Furthermore,esters production may be primarily due to CmLOX03,CmLOX12,CmLOX16 and CmLOX18,especially straight chain ester production in melon.To verify the exact roles of specific CmLOXs in the production of aroma compounds in melon,further studies on these genes should be conducted.4.YMR was used as material to investigate effects of MeJA on CmLOXs expresssions.Exogenous MeJA increased LOX activity during melon storage.Expression of CmLOX03 and CmLOX18 were increasing then decreased during 10 days storage and expressions of CmLOX03 and CmLOX18 of MeJA treatments were higher than control.CmLOX09 and CmLOX16 expressions were decareased during storage.MeJA treatment inhibited CmLOX09 expression but significantly up-regulate expression of CmLOX16.These results showed that CmLOX03,CmLOX16 and CmLOX18 expressions were positive regulated by MeJA,CmLOX09 expression was inhibited by MeJA.5.Full-length cDNAs of the oriental melon aroma relevance candidate gene CmLOX03 and its most similar gene CmLOX04 were cloned.The CmLOX03 harbours a 2634-bp open reading frame(ORF).The deduced CmLOX03 protein sequence consists of 877 amino acid residues with a calculated molecular mass of 99.634 kDa and a theoretical isoelectric point(pI)of 6.18.The CmLOX04 harbours a 2640-bp ORF encoding a predicted protein of 879 amino acid residues with a calculated molecular mass of 99.860 kDa and a theoretical pI of 5.99.Two sequence alignments of the nucleotides and deduced amino acid sequences of CmLOX03 and CmLOX04 isolated from oriental melon and from the melon genome database were done.Comparability between CmLOX03 and GLOX03 in genome database was 85.97%;comparability between CmLOX03 and LOX1(DQ267934.2),and between CmLOX03 and LOX2(GQ386815.1)was 96.47%and 99.54%,respectively.Amino acid sequences of CmLOX04 were complete same as GLOX04 in genome database.By analyzing the deduced amino acid sequences of CmLOX03 and CmLOX04 using some bioinformatics software,the PLAT and LOX domains are found and the length of the chloroplast transit peptide of the N-terminus of the deduced.Several highly conserved regions of the predicted CmLOX03 and CmLOX04 proteins are identified by amino acid sequence alignments and it is predicted that they are 13-LOXs.6.Trough Gateway technology,CmLOX03 and CmLOX04 were transferred into over-expression vectors.Using Agrobacterium-mediated method CmLOX03 and CmLOX04 were transient expressed in oriental melon.The expression level of CmLOX03 and CmLOX04 in transient expression p-CmLOX03 melon fruit were significantly up-regulated,lead to a significant increase on ethyl acetate and hexyl acetate accumulation.LOX activities in transient expression melons were not significant different with control.These results indicate that straight chain esters biosynthesis maybe depends on CmLOX03 and CmLOX04 was not involved in straight chain esters biosynthesis.