Proteomics And Fruit Quality Formation At Different Development Stages Of Oriental Melon

Oriental melon(Cucumis melo var.makuwa Makino)is one of the most popular crops for its nutritional and flavour quality,and it has a sweet and crisp taste,juicy flesh,intense volatile aromas compound and the largest plantation in china.During oriental melon ripening,components of the quality index,such as sugar,colour,texture,flavour and volatiles etc.,change significantly.At the same time,a large number of proteins and genes expression changed concomitant with ripening.Thus,correlating the proteomic profiles with the biochemical and physiological changes occurring in the oriental melon is very important for advancing our understanding of oriental melon quality in the ripening processes.The study was conducted on ’YuMeiren’ oriental melon fruit at different developmental stages.Physiological quality indices,including firmness,rind colour,soluble solids content(SSC),ethylene production,sugar content and volatile compounds were also characterized during four maturity periods of the melon,including 5,15,25 and 35 days after anthesis(DAA).We used iTRAQ to research and identify the differentially expressed proteins in melon fruit ripening and to obtain important proteins related to fruit quality.Validation the key proteins of lipoxygenase(LOX),alcohol dehydeogenase(ADH),alchol acyl transferase(AAT),sucrose synthetase(SS)and sucrose phosphate synthetase(SPS)in a-linolenic acid metabolic and starch and sucrose metabolism pathways from gene expression,enzymatic activity and Western-blot respectively.The main results are as followings:1.Four mature stages of oriental melon were employed to study the quality index.In the process of fruit ripening,firmness decreased slightly after 25 d to reach a maximum and the rind color faded green to yellow gradually.SSC and fruit weight were significantly increased to reach the maximum at 35 d.Ethylene content were significantly increased at 35d.Therefore,physiological maturity of oriental melon cultivar ’Yumenren’ was at 35 days after anthesis.2.The volatile component and sugar content were studied by GC-MS and HPLC.We identified a total of 39 volatile compounds in oriental melon during ripening,including 17 esters,12 alcohols,4 aldehydes and 4 acids.The content of aldehydes remained stable at different ripening stages.Acids decreased and content of esters and alcohols increased significantly during ripening.The main esters were ethyl acetate,butyl 2-methyl acetate and hexyl acetate.A principal component analysis(PCA)revealed that there was a negative correlation between the acids and esters.The aroma volatiles at 5 and 15 d were similar,they are acids,alcohols and aldehydes.The main aroma volatiles of 25 and 35 d are alcohols and esters,respectively.Of the three soluble sugars,fructose and glucose accumulated slowly and synchronously.In the 35 d of oriental melon,sucrose rapidly accumulated and reached its maximum,3.We used iTRAQ to research and identify the differentially expressed proteins in melon fruit ripening.More than 5835 proteins were identified and quantified and divided into 4 clusters by hierarchical cluster analysis.We pairwised compare proteins in these four stages and screen out 1694 differential proteins.We made a clustering analysis on these differential proteins and divided them into four categories,respectively represented the proteins reaching the peak on the 35,25,15 and 5 d.A functional analysis was performed using Blast2GO software based on the enrichment of a GO analysis.The majority of up-regulated proteins in Cluster 1 involved biological processes are response to stress and fruit ripening and fatty acid metabolic process.While many proteins were up-regulated first and then down-regulated in Cluster 2,these proteins participated in the response to oxidative stress,osmotic stress and glycerol ether metabolic processes.The majority of proteins in Cluster 3 involved in hormone-mediate and the root development process.While many down-regulated proteins in Cluster 4 participated in DNA repair,rRNA processing and the mRNA splicing process.4.Using KEGG database information to study the relationship between different proteins of biological pathways and finally we screened of LOX,ADH,AAT,SS and SPS from glycolysis,a-linolenic acid and starch and sucrose metabolism pathways.The results showed that two different kinds of LOX protein expression have different variety rules during melon ripening.13-LOX increased first and decreased slightly after 25 d.9-LOX,ADH,AAT,SS and SPS increased significantly in the ripening of oriental melon.5.Expression analysis was conducted for genes by RT-qPCR to further verify the results of proteomics.The results showed that 18 CmLOXs were constitutively expressed but varied greatly in the different ripening stages.CmLOX06,CmLOX10-15 and CmLOX18 were similar to the protein expression pattern of 13-LOX.CmLOX07-09,CmLOX16 and CmLOX17 were similar to the protein expression of 9-LOX.In our study,except for CmAAT3,the expression levels of CmAAT1,CmAAT2 and CmAAT4 were synchronized with the quantity change in proteins.Gene expression of CmSSl and CmSPSl increased significantly during ripening and were synchronized with the quantity change in proteins.6.Enzyme activities and Western-blot were conducted fo to verify the differentially expressed proteins by polyclonal antibody preparation of CmLOX09,CmLOX18,CmADH2,CmAATl,CmSSl and CmSPSl.The result showed that the enzyme activities of LOX,ADH,AAT,SS and SPS have same change rules with the corresponding proteins in four ripening stages.The protein expression of CmLOX09 remained stable during fruit ripening,CmLOX18 increased significantly in the 25 d,which was similar to the protein expression of 13-LOX.The protein expression of CmADH2 was not high and increased slightly after 25 and 35 d,which was similar to the protein expression of ADH by iTRAQ.CmAAT1,CmSS1 and CmSPS1 were similar to the protein expression pattern of the corresponding proteins in iTRAQ.