The Role Of Ethylene In Phenolic Biosynthesis Of Fresh-cut Pitaya Fruit

Fresh-cut fruit and vegetables have been gradually become an essential part of fruit and vegetables consumption owing to their freshness,convenience,health,nutrition and other advantages.However,minimally processed fruit is more prone to quality deterioration than intact ones,which will lead to serious losses.At present,the main factor limiting the development of new preservation technology for fresh-cut fruit and vegetables is the lack of deep understanding of the key basic theories of wounding stress responses.Phenolic compounds are important antioxidants in fruit and vegetables,which play an important role in wounding response.The biosynthesis and accumulation of phenolic compounds to wounding stress is one of the most significant responses in multiple fruit and vegetables.Previous studies have shown that the phenolic biosynthesis is closely related to the regulation of ethylene signaling molecule,but most of the relevant studies focused on the direct regulation of ethylene signal on the terminal reaction of phenolic accumulation,and the transcriptional regulation of ethylene signal on phenolic biosynthesis still needs further investigation.Ethylene response factors(ERFs)are key transcription factors at the end of ethylene signaling pathway that play an important role in plant wounding responses and biosynthesis of secondary metabolites,but the transcriptional regulation mechanism of ERFs involved in the phenolic biosynthesis of fresh-cut fruit and vegetables remains unclear.In this paper,pitaya fruit was taken as experimental material and the role of ethylene signaling pathway in the phenolic biosynthesis of fresh-cut pitaya fruit was studied.Firstly,the key transcription factors that may regulate the phenolic biosynthesis of fresh-cut pitaya fruit such as HuERF1 were screened by bioinformatics analysis.Then the role of ethylene in the phenolic biosynthesis of fresh-cut pitaya fruit was clarified by pretreatment with exogenous ethylene and its receptor inhibitor 1-methylcyclopropene(1-MCP).Moreover,according to the analysis of key genes involved in endogenous ethylene biosynthesis and signal transduction,it was found that the phenolic biosynthesis of fresh-cut pitaya fruit induced by exogenous ethylene was closely related to ethylene signal transduction pathway and HuERF1 played an important role in this process.Furthermore,the regulatory roles of HuERF1 on target genes in the phenylpropanoid metabolism were analyzed according to the electrophoretic mobility shift assay(EMSA)and dual luciferase reporter assay(DLR).The main results were as follows:(1)Bioinformatics analysis of key regulatory genes in phenolic biosynthesis of fresh-cut pitaya fruit.The changes of total phenolics content and signal substances such as ethylene in fresh-cut pitaya fruit at the initial,early,middle and end of storage(W0h,W6 h,W24h,W48h)were analyzed and verified.Weighted gene co-expression network analysis(WGCNA)and other bioinformatics methods were used to reveal the changes of key genes involved in the phenolic biosynthesis systematically,and key transcription factors such as HuERF1 which may regulate this process were filtered.The results indicated that the phenolic biosynthesis was closely related to the gene regulation of primary metabolism,secondary metabolism and signal transduction pathways such as ethylene,and transcription factors including HuERF1 may be involved in the transcriptional regulation in this process.(2)The effects of ethylene and 1-MCP pretreatment on the biosynthesis and accumulation of phenolics in fresh-cut pitaya fruit were studied.The results found that the gene expressions and activities of key enzymes(phenylalanine ammonia lyase(PAL),cinnamic acid 4-hydroxylase(C4H),4-coumarate-Co A ligase(4CL))closely related with phenolic biosynthesis were promoted by ethylene pretreatment,and the accumulation of total phenolics and flavonoids,phenolic compounds such as chlorogenic acid,gallic acid,protocatechuic acid,phloridzin and kaempferol were also improved by ethylene pretreatment in fresh-cut pitaya fruit,which led to the enhancement of antioxidant capacity.But 1-MCP pretreatment inhibited the activities and gene expressions of PAL,C4 H and 4CL,and delayed the accumulation of phenolics as well as the enhancement of antioxidant capacity in fresh-cut pitaya fruit.The results indicated that ethylene was involved in the biosynthesis and accumulation of phenolics in fresh-cut pitaya fruit by regulating the key enzyme activities and gene expressions of phenylpropanoid metabolism pathway.(3)The effects of ethylene and 1-MCP pretreatment on ethylene signal transduction pathway of fresh-cut pitaya fruit were investigated.The results found that ethylene pretreatment suppressed the wound-induced production of endogenous ethylene,but effectively improved the up-regulation of the relative expressions of key ethylene receptor genes(HuETR1 and HuETR2)and their downstream transcription factor genes(HuEIN3 s and HuERF1s),and further reduced the down-regulation of the negative ethylene regulator(HuCTR1s)caused by wounding stress.While the application of 1-MCP had no significant effect on endogenous ethylene biosynthesis,but negatively regulated the relative expressions of HuETR1 and HuETR2,suppressed the wound-induced up-regulation of HuEIN3 s and HuERF1 s,and weakened the downregulation of HuCTR1 s.The results demonstrated that exogenous ethylene induced the biosynthesis and accumulation of phenolics in fresh-cut pitaya fruit by regulating the ethylene signal transduction pathway,and the terminal transcription factor HuERF1 played an essential role in this process.(4)The regulatory effects of HuERF1 on the target genes in the phenolic biosynthesis of fresh-cut pitaya fruit were studied.The full-length CDS sequence of HuERF1 and the promoters of key genes in the phenylpropanoid metabolism pathway(HuPAL,HuC4 H and Hu4CL)were cloned based on the pitaya genomic database.Further EMSA and DLR assay indicated that HuERF1 could specifically activate the transcription of HuPAL and HuC4 H by binding to GCC-box elements in their promoters,but no targeted regulation of HuERF1 on Hu4 CL was found.