Study On The Molecular Mechanism Of Aroma Synthesis Regulated By Ethylene In Cold Storage Peach Fruit

Peach belongs to climacteric fruit, with fruit spoilage after3-5d storage at roomtemperature. Cold storage is the main method to extend the market supply of peach fruit. Whilepeaches are coldsensitive fruit and prone to chilling injury during cold storage. Fruits Chillinginjury syptoms show up as flesh browning, innormal soften and cotton, and low juice yield etc..Low temperature resulted in the loss of flavor peach fruit, which seriously affected the edibilityof the fruits. Early research of our laboratory showed that the content of Floral/Fruity aromavolatiles were significantly lower than which stored at the ambient temperature, while thecontent of Green aroma volatiles were higher. The aroma volatiles in cold storage peach fruitswere to some extent promoted after treated by exogenous ethylene. The release quantity ofethylene significantly reduced under low temperature. On the other hand, the activity of ACOenzyme reduced. While the opposite result was received from the fruit treated by exogeneousethylene. There was necessary connection between ethylene and fruit aroma-volatile-synthesis.But till now, the molecular mechanism on synthesis of aroma volatile regulated by ethylene isunclear. Metabolites are usually positively correlated with metabolic substrates. So it isnecessary to research the regulation of ethylene on aroma synthesis of peach fruit from thegene transcription level, combining with the aroma volatile metabolite profiling. The mainresults of this research are as following:1. Cold storage has a greate impact on gene expression in peach fruit. Compared withCK1, expression level of genes related to energy metabolism and pathogenesis-related proteingenes such as ATPase, cryptdin, thaumatin and Chitin were greatly upregulated in CK2. Thismaybe is stress response to low temperature. After treated by exogenous ethylene, theexpression of these genes were significantly downregulated, but which were still higher than T.This result reveled that the transcriptional expression of these genes were co-regulated by low temperature and ethylene. Metabolic disorders of energy and resistance-related is the reason offruit chilling injury at low temperature storage.2. As far as CK2sample, the expression levels of stearoyl CoA thioesterase gene involvedin stearic acids synthesis were significantly downregulated even no expression. Exogenousethylene does not change the expression levels of stearoyl CoA thioesterase genes. In CK2, themain unsaturated fatty acids substrates for synthesis of aroma volatiles were linolenic/linoleicacid because of transcriptional inhibition of key genes involved in stearic acid synthesis. Underlow temperature, linolenic/linoleic acid CoA thioesterase genes were upregulated. Metabolicdisorder of stearic acids leaded to lack supply of substrate in cold storaged peach fruit maybethe reason that some aroma volatiles metabolic disorder.3. LOX genes involved in the synthesis of aldehydes aroma substances via α-linolenicacid pathway. Different family members of LOX, ALDH, EH may be involved in differentbiological processes and they have different sensitivity to ethylene pathway through analysis oftheir expression patterns. Ethylene response factor binding protein transcription factor ERF12is a transcription repressor, positively regulated by ethylene. The highest expression level ofERF12was in CK1sample, which downregulated under lower temperature, and upregulatedafter treated by ethylene. Ethylene response factor binding protein ERF1, ERF2, ERF5astranscriptional activators negatively regulated by the ethylene, which the lowest level ofexpression of the CK2. Under low temperatures, the expression of ERF12gene downregulated,lifting the inhibition of transcription for LOX gene. In peach fruit T sample, the expression ofLOX family genes downregulated after treated by ethylene because of the inhibitiontranscription of ERF12. Ethylene regulated synthesis of green aroma volatiles such as hexanal,trans-2-hexenal, trans-2-hexenol and hexenyl acetate and fruity aroma volatiles such as hexylacrylate, o-aminobenzaminicacid-1,5-dimethyl-1-ethylene-4-hexenyl and dibutyl glutaratethrough transcriptional activition or repression of LOX, ADH, AAT, ALDH controlled by ERF1,ERF2, ERF5and ERF12. 4. Lipoxygenase gene was cloned from peach fruit by homologous cloning and RACEtechnology. The gene belong to LIPOXYGENASE superfamily with full length2640bp,3’UTR269bp and encoding879amino scids. The deduced amino acid sequence contains twoconserved domains: PLAT/LH2and Lipoxygenase. The result of real-time quantitative PCRshowed that the expression of this gene significantly downregulated in CK2than CK1andwhich was higer than in T. This result was consistent with the result of DGE sequencing. Total14LOX homologous genes were retrieved from okubo fruit with two clusters afterphylogenetic analysis:13-LOX and9-LOX. Five genes were not detected by DGE. There werefour of the nine genes negative regulated by ethylene respectively as PpLOX4, PpLOX10,PpLOX11and PpLOX12.5. Epoxide hydrolase gene was cloned by homologous cloning and RACE technologywith1274bp full length,161bp and143bp5’UTR and3’UTR respectively, and with972bpORF. This gene encoded323amino acids. The deduced amino acid sequence are αβ hydrolase/folding enzymes configuration with a trinity of Asp-His-Asp activation center and Y-Y capstructure. The protein was located in the chloroplast. The result of Real-time PCR analysisindicated that this ge ne was co-regulated by low temperature and ethylene. It maybeparticipate in a number of different physiological and biochemical processes.6. Alkanes were the high level volatile ingredients from ‘okubo’ peach fruit in relativecontent. The alkane were oxidized to1-alcohol by CYP716A1.Then the aldehydes and alcoholsconverted into each other under the activition of ADH. The substances involved in thesynthesis of alkanes were positively controlled by ethylene. Alkanes participated in theaccumulation and release aldehydes and alcohols from peach fruit by providing a substrate forsynthesis of1-ol. Accumulation of alcohols were effected by the linkage of alkane-1-monooxygenase CYP716A and ALDH and the linkage of CYP716A and epoxide hydrolaseEH respectively through fatty acid degradation pathways. The transcription expression level ofGDSL gene changed with changed in fatty acid levels and was to maintain the homeostasis of fatty acid in organ body.