Molecular Mechanism Of Nitric Oxide Regulation On Alleviating Chilling Injury In Peach Fruit During Storage

[Objective]Peach is a typical climacteric fruit.Postharvest peach fruit is easy to soften and ripen at room temperature,resulting in shorter storage period.Low temperature storage is beneficial to the freshness of postharvest peach fruit.However,peach fruit is sensitive to cold temperature and is susceptible to cold damage symptoms under long-term cold storage.At the same time,the high levels of reactive oxygen species（ROS）generated by low temperatures can cause oxidative damage and result in mitochondrial dysfunction.Therefore,to explore the mechanism of maintaining the functioal integrity and alleviating oxidative damage of mitochondria is of great significance for keeping fruit storage quality and delaying fruit aging.Some studies have shown that nitric oxide（NO）is an important signal molecule involved in the response to biological and abiotic stresses and plays a regulatory role in the maturation and senescence,and the mitigation of storage chilling injury for plants.At present,the research on the oxidative damage of NO to the mitochondria mainly was focused on the regulation of the antioxidant enzyme system of cold fruit mitochondria,and there are few studies on whether it has a regulatory effect on the function and gene replication and transcription of plant mitochondria.DNA methylation is a significant genetic modification in response to abiotic stress in plants.This study aimed to explored the effects of NO on mitochondrial function and mitochondrial gene replication and transcription in peach fruit during cold storage,as well as the relationship between NO and DNA methylation,to providing a theoretical basis for alleviating oxidative damage from low temperature and chilling injury to peach fruit storage,and improving the cold resistance of cold peach fruit.[Methods]‘Feicheng’peach fruit was used as test material and were soaked in NO solution(15μmol L^-1),c-PTIO(20μmol L^-1,carboxy-PTIO,NO scavenger)solution,and double-distilled water（control）for 30min,stored at 0°C for 5 weeks after the treatment.The fruit quality,mitochondrial function and antioxidant system related indicators of cold peach fruit were analyzed to study the regulatory effect of exogenous NO treatment on mitochondrial function and oxidative damage.The mitochondrial transcription termination factor m TERF18 gene of peach fruit was cloned by RT-PCR technology,and PpmTERF18gene was overexpressed in tomato and peach fruit by Agrobacterium-mediated transient transformation,and the role of PpmTERF18 in fruit mitochondrial function and antioxidant capacity was tested.The regulatory effect of NO on DNA methylation in cold peach fruit was detected with bisulfite sequencing PCR（BSP）technology.[Results]（1）By Exogenous NO treatment,the endogenous NO content were significantly increased,and the browning and softening of cold peach fruit were delayed.Maenwhile mitochondrial ROS content was significantly inhibited because the activity of enzymes related to ROS metabolism was improved,fourthermore the oxidative stress caused by Low temperature was alleviated on peach fruit.Exogenous NO treatment significantly inhibited the decrease in membrane fluidity and mitochondrial respiratory control rate,and suppressed the opening of mitochondrial permeability transition pore（MPTP）and mitochondrial oxygen consumption,so maintained mitochondrial function.Exogenous NO treatment significantly up-regulated the expression of PpmTERF18 and increased mitochondrial DNA copy number in peach fruit,indicating that NO could maintain the stability of mitochondrial DNA（mt DNA）by regulating mitochondrial copy number.（2）The peach fruit m TERF18 protein is composed of 573 amino acid residues,with a molecular weight of64996.38.It is a hydrophilic protein without a secretory signal peptide.Secondary structure prediction of the protein revealed that theα-helix and irregular helix in the peach fruit m TERF18 protein are the main structural elements in the overall protein structure.The sequence of the peach fruit m TERF18 gene was conserved and showed high sequence similarity to those of almond,sweet cherry,white pear,apple and moonflower.Tobacco subcellular localisation results showed that it is localised in mitochondria.Gaussian software simulations showed that Cys 281 had the best reactivity and stability in the glutathionylation reaction between NO and PpmTERF18,and Tyr 300 had the best stability and reactivity in the nitrosylation reaction between NO and PpmTERF18.（3）Overexpression of PpmTERF18 in tomato delayed the colour change of tomato fruit;and overexpression of PpmTERF18 in peach fruit maintained the quality of peach fruit.Overexpression of PpmTERF18 significantly increased the activity and gene expression of mitochondrial ROS metabolism-related enzymes in tomato and peach fruit;overexpression of PpmTERF18 significantly reduced the content of O₂^·-,H₂O₂and?OH in mitochondria of tomato and peach fruit,decreased ROS production,improved the defense capacity of antioxidant system and reduced oxidative damage in tomato and peach fruit;overexpression of PpmTERF18 significantly inhibited the oxidative damage in tomato and peach fruit.Overexpression of PpmTERF18 significantly inhibited the reduction of mitochondrial membrane potential,mobility and respiratory control rate,inhibited the opening of MPTP,reduced mitochondrial oxygen consumption and cytochrome c content,increased mt DNA copy number and reduced mt DNA damage to maintain mitochondrial function in tomato and peach fruits.Overexpression of PpmTERF18 significantly increased the content of total phenols and total flavonoids,reduced the content of MDA and maintained membrane stability in peach fruit,indicating that overexpression of PpmTERF18could maintain fruit quality.（4）Exogenous NO significantly up-regulated the total DNMT activity and the expression of methylase-related genes;NO treatment significantly up-regulated the expression of Pp CBF5,Pp ICE1,Pp MYC2,and Pp COR in cold peaches,and the expression of NO-mediated promoter region DNA Methylation in response to cold stress induces changes in transcriptional levels of cold tolerance transcription factors（TFs）in peach fruit.Further BSP analysisof Cp G island and promoter regions of four cold resistance genes showed that NO signal induced increased methylation of Pp CBF5,Pp ICE1,and Pp MYC2 promoters and demethylation of Pp COR promoter regions.Different Cp G islands of Cp G have different responses to NO regulation,and NO can modify cytosine C methylation sites.Cold-resistant transcription factors are actively involved in the process of cold stress.These results indicated that NO,cold stress,and DNA methylation are closely related,explaining that NO enhances the cold tolerance of peach fruit by mediating DNA methylation.Exogenous NO significantly up-regulated total DNMT activity and the expression of methylesterase related genes;NO treatment significantly up-regulated the expression of Pp CBF5,Pp ICE1,Pp MYC2 and Pp COR in chilled peaches,and NO-mediated DNA methylation in the promoter region in response to cold stress,thereby inducing changes in the transcript levels of cold-tolerant transcription factors in peach fruit.Further bisulfite sequencing（BSP）analysis showed that Cp G islands of four cold-tolerant genes and NO signalling induced methylation of the promoter regions of Pp CBF5,Pp ICE1 and Pp MYC2 and demethylation of the Pp COR promoter region,indicating that different Cp G islands in the promoter regions responded differently to NO regulation and that NO could modify the cytosine C methylation site.These results suggest that NO participates in the process of cold stress by regulating the expression and methylation level of cold tolerance transcription factors,cold stress and DNA methylation are closely related,explaining that NO enhances cold tolerance in peach fruit by mediating DNA methylation.[Conclusion]The scavenging capacity of reactive oxygen species in mitochondria of cold peach fruit was significantly improved,cell membrane integrity and function were maintained,and oxidative damage was reduced during low temperature storage of peach fruit treated by Exogenous NO;m TERF18 protein in peach fruit was localized in mitochondria and consisted of 573 amino acids,unstable hydrophilic protein without secretory signal peptide and transmembrane structure.Cys 281 showed good stability and reactivity with NO in response to glutathionylation.Overexpression of PpmTERF18 significantly increased the defence capacity of the mitochondrial antioxidant system and maintained mitochondrial function in tomato and peach fruit.Transcript levels of cold-tolerant transcription factors in cold peach fruit mediated by NO was increased so the cold resistance of cold peach fruitand was improving.It was revealed that exogenous NO enhanced cold tolerance in cold peach fruit by improving the defence capacity of the mitochondrial antioxidant system and mediating DNA methylation,and improved storage quality and alleviated cold damage in cold peach fruit.