Studies On Postharvest Of Apple Superficial Scald And Litchi Pericarp Browning

Laccases(p-diphenol:O₂-oxidoreduacatse,E.C 1.10.3.2)have been found in plants and can catalyze the oxidation of a large range of substrates.Superficial scald often develops during warm temperature storage following removal from cold storage,or long-time cold storage.The superficial scald symptoms are similar to Litchi pericarp browning,reducing apple commercial value.Although a lot of work were investigated,the mechanisms leading to brown color formation in fruit pericarp tissue are not completely known.Many studies suggest that diphenylamine(DPA)and 1-methylcyclopropene(1-MCP)treatment can effectively control the formation of superficial scald during apple storage,but the effects of these treatments on laccase and secondary metabolites have not been reported.In the present on apples,two scald-susceptible apple cultivars,‘Red delicious'and‘Cortland',were investigated;we combined LC-MS with non-targeted metabolomics techniques to explore the correlation between metabolomics and postharvest of apple superficial scald,then the laccase genes in apples was cloned and characterized,to study gene expression of laccase genes and its enzyme activity in response to DPA and1-MCP treatments,and to examine changes in phenolic compounds in response to those treatments.Based on the evidences collected from this study,we developed a hypothesis that laccase may also contribute to superficial scald development in apples.The biological significance of laccase and metabolism in apples is discussed within the context of apple fruit scald development and treatments.Litchi(Litchi chinensis Sonn)is a typical non-climacteric fruit,the rapid postharvest browning of its pericarp tissue limits the shelf life of the litchi,reducing the commodity value of the fruit.Our research showed that laccase is a key enzyme in litchi browning by catalyzing the synergistic reaction between epicatechin and anthocyanin.Therefore,we analyzed the accumulation of laccase substrates and expression patterns of related genes during the development of litchi fruits.It will provide more evidence to understand the mechanism of litchi browning.In addition,Studies have shown that loss water can lead to litchi pericarp browning and increase laccase activity.The aerial parts of litchi pericarp are covered with a cuticle,a hydrophobic layer consisting of cutin polyester and cuticular waxes that protects them from water loss.In this study,we studied the relationship between litchi pericarp browning and epidermal wax,then the function of Lc WAX2 was further studied.The main results were outlined as follows:1.Study on metabolomics of apple superficial scald during the storageAmong the metabolites,545 compounds were detected using LC-MS with untargeted mode of Waters q TOF during the development of superficial scald in apple.However,57were tentatively identified so far.From the identified compounds,ten metabolites were significantly responded to treatment of DPA and 1-MCP.They were glyceric acid,cinnamaldehyde,6-methyl-5-hepten-2-one,2,4-dihydroxycinnamate,cinnamyl cinnamate,methylarbutin,5,7,4'-trihydroxyflavanone-7-O-arabinosylglucoside,quercetin-3-rutinoside,3,2',4',6'-tetrahydroxy-4-methoxy-3',5-diprenyldihydrochalcone,rhamnetin-3-neohesperidos ide,respectively.PCA analysis indicated the significant interaction among cultivars,treatments and storage in this study,which shows 72%variation of the analysis data.The right up corner were the markers which seem to be associated with scald development.They were 6-methyl-5-helpten-2-one,3-O-beta-D-Galactopyranosylproanthocyanidin A5',4-O-caffeoylquinic acid,quercetin-3-rutinoside,rhamnetin-3-neohesperidoside,cyanidin-3-diglucoside-5-glucoside etc.We also identified some interesting compounds such as 6-methyl-5-helpten-2-one,3-O-beta-D-Galactopyranosylproanthocyanidin A5',4-O-caffeoylquinic acid,quercetin-3-rutinoside,rhamnetin-3-neohesperidoside,cyanidin-3-diglucoside-5-glucoside which were increased in control in both cv.‘Cortland'and cv.‘Red delicious'fruit,but decreased response to DAP and 1-MCP treatments,which may play a role in apple superficial scald development.Our LC-MS results indicated that laccase may react with 4-O-caffeoylquinic acid as substrate,this is new finding that it may be the substrate for laccase in apples.2.Study on the role of laccase and phenolic compounds during the developement of apple superficial scaldThe full-length complementary DNAs encoding laccase genes were obtained from‘Red delicious'and‘Cortland'fruit.The apple Laccases were compared to those in other plant species and showed up to 99%homology to Arabidopsis and Litchi.q RT-PCR analysis revealed changes in transcript abundance of certain Md LAC genes(2,7,9,12,14,15 and 16)during storage and in response to DPA and 1-MCP treatments.Significant increases in laccase gene expression of Md LAC7 were found in untreated apples during storage from 4 to 7 months in‘Cortland'and‘Red delicious'(especially by day 7 at 20 ^oC following seven months cold storage),while Md LAC12,14,15 and 16 decreased in‘Red delicious'.Meanwhile,gene expression levels of Md LAC7,9,12 and 14 in‘Cortland'and Md LAC7,9,12 and 14 in‘Red delicious'were significantly lower for both DPA and 1-MCP treatments,despite the fact there were no changes in expression between four and seven months of storage.No significant change in expression of Md LAC12,Md LAC15 and Md LAC16 was found for‘Cortland'during the storage,neither in response to DPA nor1-MCP treatments when compared with the control.Enzyme activity of laccase protein in apple peel increased with storage in control fruit,while decreasing significantly with DPA or 1-MCP treatment.Concentrations of phenolic compounds in pericarp tissues decreased generally during storage,except for chlorogenic acid,which is increased.An increase of phlorizin,rutin equivalents and cyanidin-3-glucoside equivalents induced by DPA treatment at four months was detected.Among the phenolic compounds,epi-catechin was higher in both cultivars as compared with catechin and chlorogenic acid.But no significant changes in catechin,epi-catechin and chlorogenic acid in response to DPA and 1-MCP treatments was found.PCA analysis showed that apple superficial scald was negatively correlated with phlorizin and positively correlated with chlorogenic acid,but positively correlated with catechin in‘Red delicious'fruit.These results indecated that laccases and phenolic compounds in apples may play an important role in superficial scald development during storage.3.Purification and accumulation of substrates of laccase in litchi pericarp browningThe identified proanthocyanidins and catechins were detected in the pericarp of the young fruit,and the levels of these compounds decreased as the fruit developed,correlating to the decreasing expression of Lc LAR and Lc ANR,two key genes in the catechin biosynthesis pathway.Lc LAC level was highly detected in young fruit and low level in mature fruit.Laccase can catalyze the reaction of epicatechin and form condensed tannins.Litchi pericarp extraction was purified by LH-20 and obtained four fractions,P1–P4.Using HPLC-Q-TOF-MS/MS,one B-type and complex A/B type epicatechin trimers were identified in P3;another B-type and two A/B-type trimers were identified in P4.P1 and P2,containing epicatechin and proanthocyanidin B2,respectively.It is the first time that the two B type trimers of epicatechins(Litchitannin B1 and B2),have been found in Litchi species.These results indecated that epicatechin oligomers may be the substrate for laccase.4.Study on the relationship between wax synthesis and Litchi percicarp browningOur research showed that loss water can lead to litchi pericarp browning and increase laccase activity.The aerial parts of litchi pericarp are covered with wax layer,which protects them from water loss.But the relationship between wax and litchi pericarp browning has not been reported.The total wax was mainly accumulated at 60 days after anthesis,but the level of‘Guiwei fruit'was higher than‘Nuomici'fruit.Scanning electron microscopy(SEM)of pericarp structure showed that heavier wax deposits on the cuticular layer of litchi fruit at 60 days after anthesis.The parallel cuticular lines formed a network in cv.Guiwei,and intercellular ridges with more wax deposits were observed.The cuticular layer of cv.Nuomici had a protruding‘raised'structure with fewer wax deposits.The physiological indices showed that indices for water loss,total wax and browning index of cv.Nuomici decreased more than those of cv.Guiwei during storage.These results indicated that cv.Nuomici had a short shelf life,and that package treatment could delay the browning of litchi pericarp.qRT-PCR analysis revealed the patterns of the expression of Lc CER1 and Lc WIN1,correlating to the change of the total wax.Lc CER1,Lc WAX2 and Lc WIN1 may play an important role in the wax synthesis in Litchi.Where the weight loss of litchi pericarp was significant,significant increases in wax-related genes expression of Lc KCS,Lc ECR,Lc LACS,Lc WAX2 and Lc WIN1 were found in untreated litchi during storage.We reported for the first time that the expression of wax-related genes,Lc KCS,Lc ECR,Lc LACS,Lc WAX2 and Lc WIN1 were sensitively responding to water loss during fruit storage,these wax-related genes may delay the water loss and litchi pericarp browning.5.Study on the function of Lc WAX2 gene in litchi pericarpClustal X and MEGA6 analysis showed that the Lc WAX2 protein has six transmembrane domains.Full-length Lc WAX2 showed high identity to Rc WAX2 with99.57%.Subcellular localization showed that Lc WAX2 protein was localized to the endoplasmic reticulum.To understand further the function of Lc WAX2,p B2GW7-Lc WAX2vector was constructed and then transformed into tomato and two transgenic lines were obtained.Two transgenic tomato lines(OX-Lc WAX2-1 and OX-Lc WAX2-2)were confirmed by q RT-PCR.Compared with wild-type tomatoes,the abundance of Lc WAX2 transcript was 8 and 4.5 times and total wax was 1.94 and 1.78 times in OX-Lc WAX2-1 and OX-Lc WAX2-2,respectively.GC-MS showed that epicuticular wax was mainly composed of alkanes,aldehydes,ketones,primary alcohols,fatty acids and so on.Among the several wax component,alkanes were 2.27 and 2.07 times in OX-Lc WAX2-1 and OX-Lc WAX2-2respectively,when compared with the wild-type.Overexpression Lc WAX2 decreased the weight loss rate of tomato fruit.These results indicated that Lc WAX2 plays a pivotal role in alkanes biosynthesis and litchi pericarp browning.