Mitochondrial Energy Metabolism And Reactive Oxygen Species Involved In Early Defense Responses Against Pink Rot And Si Induced Resistance Of Harvested Muskmelons

Trichothecium roseum is a major postharvest fungus which causes pink rot of harvested muskmelons in Northwest China.Sodium silicate?Si?is an inorganic elicitor of plant.Our previous research indicated that muskmelons defensed infection of T.roseum and induced resistance by Si were related with the energy metabolism and reactive oxygen species?ROS?.However,the mechanism in detail is not clear.In this study,the harvested muskmelons?Cucumis melo L.cv.Yujinxiang?were used as material.Based on isolating and purifying of fruit mitochondria,we investigated the effects of T.roseum infection and Si treatment on energy metabolism and ROS production of fruit.The mitochondrial proteomics was also analyzed on infected and treated muskmelons.The results indicated as below:1.A two-step Percoll density gradient differential centrifugation was used to establish a reliable method for isolation and purification of mitochondria from muskmelon fruits.The structure of mitochondria showed intact and with good activity.The phenol extraction was screened as an optimum method for extracting mitochondrial proteins from mitochondria when compared with other extraction methods.2.T.roseum infection obviously induced H₂O₂ accumulation in mitochondria.Enzyme activities were inhibited in the first 6 hours post inoculation?hpi?,including succinic dehydrogenase,cytochrome c oxidase,H⁺-adenosine triphosphatase?ATPase?and Ca²⁺-ATPase.However,the activities of Ca²⁺-ATPase and H⁺-ATPase and the content of intracellular ATP were improved to a higher level at 12 hpi.3.A total of 1613 mitochondrial proteins in T.roseum inoculated muskmelon fruits were identified by the tandem mass tags?TMT?-based proteomics approach.A total of42 differentially expressed proteins were identified through the proteomic analyses,which were mainly involved in energy metabolism,stress responses and redox homeostasis,glycolysis and tricarboxylic acid cycle,transporter and mitochondria dysfunction.4.Si treatment at 100 mM caused lower decay incidence and severity of inoculated muskmelons with T.roseum than the control.Si induced the accumulation of hydrogen peroxide and promoted the generation rate of superoxide anion.The co-localization of fluorescent signals confirmed that mitochondria were involved in Si and pathogen induced ROS burst at 24 hours after the treatment.In addition,the activities including H⁺-adenosine triphosphatase,succinic dehydrogenase and cytochrome c oxidase were also higher in Si-treated muskmelons than those in the control at different times after inoculation with T.roseum.Si treatment also kept the intracellular ATP at higher level after inoculation.5.A total of 1694 mitochondrial proteins in the inoculated muskmelon after Si treatment were identified by the iTRAQ-based proteomics analysis.There were 51significantly altered proteins were found to lacated in mitochondrial.The proteins mainly involved in respiratory chain,tricarboxylic acid cycle,glycolytic process,oxidation reduction process,defense and stress responses,mitochondria carriers,protein and amino acids metabolism.It is suggested that mitochondria play crucial roles in the early defense responses of muskmelons against T.roseum infection through regulation of ROS production and energy metabolism.Moreover,as a safe and effective elicitor,sodium silicate mobilizes mitochondrial ROS and energy metabolism against T.roseum infection in a stronger,speeder,and more sustainable manner.