Integrated Transcriptome And Metabolome Analysis Of Post-harvest Apples Response To Mechanical Damage

The mechanical forces such as compression and impact that fruits suffered during the supply chain are the main factors leading to mechanical damage.Apple(Malus×domestica Borkh.),a fruit of the Rosaceae family,is an important bulk fruit in China and is well popular with consumers.However,due to the thin peels and brittle texture of apple fruit,it is prone to mechanical damage during post-harvest logistics,leading to rot and deterioration,which significantly affects the merchantability and market competitiveness.There is a lack of systematic bioinformatics studies on post-harvest mechanical damage stress-induced changes in genes and metabolic regulatory networks in fruits.In this study,we performed integrated transcriptome and metabolome analysis of post-harvest apples subjected to compression and impact damage in'Red Fuji'apples(Malus×domestica Borkh.Red Fuji)using transcriptomic,widely targeted metabolomic and gas chromatography-ion mobility spectrometry(GC-IMS)techniques,and screened candidate defense genes and target compounds for quality monitoring,elucidated the metabolic regulatory network of mechanical damage stress.The results of the study provide theoretical support for the stress mechanism of post-harvest mechanical damage to fruits,and also provide a new path and basis for improving freshness preservation and damage reduction technologies in logistics,improving the post-harvest nutritional quality of fruits,cultivating new germplasm with high quality and storage resistance,and developing online monitoring system of mechanical damage to fruits during logistics.The main results are as follows.1.The gene expression of Md OFUT19,Md WRKY48,Md CBP60E,Md CYP450and Md SM-like in apples subjected to compression damage were consistently higher than those of the control group during storage.The metabolites such as procyanidin A1,Dl-2-Aminooctanoic acid,5-O-p-Coumaroyl shikimic acid and 5,7-Dihydroxy-3',4',5'-trimethoxyflavone in apples subjected to compression damage were higher than those of the control group.Transcriptome-metabolite association analysis revealed that the metabolic pathways that were simultaneously annotated with DEGs and DEMs in compression damaged fruit included the phenylpropanoid biosynthesis,phenylalanine metabolism,propionate metabolism,butanoate metabolism,arginine and proline metabolism(P-value<0.05).Analysis of DEGs and DEMs on the common annotation pathway revealed that the LOX pathway key gene Md AAT was significantly up-regulated,presumably related to the induction of volatile ester biosynthesis in fruits subjected to compression damage;meanwhile,the lignin synthesis pathway key genes including Md PAL,Md CCo AOMT,Md CCR,Md COMT,Md CAD,and Md PAL,were significantly up-regulated and their contents increased in response to coumarin,suggesting that the fruit may respond to compression stress by promoting lignin synthesis.The above results suggest that apple fruits exhibit resistance mechanisms in multiple metabolic pathways including LOX pathway and lignin synthesis pathway in response to compression stress.2.The expression levels of 124 genes in apples subjected to impact damage were consistently higher than those of the control during storage,mainly annotated in the phenylpropanoid metabolism,signal transduction pathway and carbohydrate metabolism.The metabolites such as isothankunic acid,2,4,2',4'-tetrahydroxy-3'-prenylchalcone,11-Keto-ursolic acid,succinic anhydride and trans-5-O-(p-Coumaroyl)shikimate in apples subjected to impact damage were higher than those of the control group.Transcriptome-metabolite association analysis revealed that the metabolic pathways that were simultaneously annotated with DEGs and DEMs in impact damaged fruit included the flavonoid biosynthesis,linoleic acid metabolism and ABC transport(P-value<0.05).Analysis of DEGs and DEMs on the common annotation pathway revealed that the Md AAT,Md LOX5 and six Md LOX1.5 genes in the LOX pathway;and the Md CHI,Md F3H,Md CCo AOMT and Md UGT88A1 in the flavonoid synthesis,were significantly up-regulated;these genes may be related to the resistance mechanisms of fruit subjected to impact damage.In addition,13 ABC transporter proteins were significantly up-regulated and a large number of secondary metabolites were accumulated,suggesting that ABC transporter proteins may be involved in the transport of secondary metabolites in response to impact stress in fruit.The above results suggest that apple fruits exhibit resistance mechanisms in multiple metabolic pathways including LOX pathway,flavonoid synthesis pathway,and ABC transport in response to impact stress.3.The results of DEGs commonality analysis of compression and impact damaged apples during storage indicated that Md WRKY48,Md CBP60E,Md CYP450 and Md SM-like might be key genes induced by mechanical damage;meanwhile,among the shared DEMs caused by both mechanical damages,the impact group showed significant higher Log₂FC of procyanidin A2 and S-Allyl-L-cysteine than those of compression group.Eight shared metabolic pathways were present among the 20 major KEGG pathways in both treatment groups.The main conclusions from the joint analysis of DEGs and DEMs in the two mechanical damage treatment groups included:the accumulation of maslinic acid in apples sufferring mechanical damage may be affected by the synergistic expression of Md CYP450 and Md SM-like;the synthesis of secondary metabolites such as flavonoids,lignans and coumarins,tannins,and terpenoids may be associated with the up-regulated expression of Md CYP450.The accumulation of sinensetin may be transcriptionally regulated by Md WRKY48;Md CBP60E may play an important role in the stress mechanism of fruit subjected to mechanical damage.4.GC-IMS technology was used to analyze the change pattern of volatile compounds in apples caused by compression damage during storage,and screened two characteristic volatile compounds(hexanal and ethyl acetate),and pointed out that they could be used as candidate targets for postharvest mechanical damage detection in fruits,and then proposed a model of online monitoring system for detecting mechanical damage.Then,the identification of genes related to the synthesis of characteristic volatiles was carried out in combination with transcriptome analysis.The results showed that the Md AAT,Md LOX-like,Md LOX3b,Md LOX7b,Md LOX7c and Md LOX2a related in LOX pathway were up-regulated expressed;1 MYB,5 ERF and5 WRKY TFs were also up-regulated expressed.Correlation analysis showed that Md AAT was significantly correlated with Md MYB-like,Md WRKY23,Md WRKY71,Md ERF15 and Md ERF2(R?0.97).In addition,Md AAT was highly expressed and had the highest transcript levels(7.13 folds higher)in damaged apples after 2 d of storage when compared with the CK group.Therefore,it is speculated that Md AAT may be regulated by transcription factors such as MYB,ERF,and WRKY during the synthesis of characteristic volatiles in fruits induced by compression damage.