| Papaya?Carica papaya L.?is easy to soften and decay after harvest with a short shelf-life.1-MCP?1-Methylcyclopropene?treatment and low temperature storage can significantly delay the softening and ripening of papaya fruit.However,improper use of 1-MCP?concentration and time?or low temperature may lead to fruit ripening disorder?no normal softening or yellowing?,which can seriously affect the quality and commercial value of papaya fruit.In this work,the effects of 1-MCP treatment and low temperature stress on the physiological and biochemical characteristics of papaya fruit,such as ripening quality and cell wall metabolism were studied.Then the expression patterns of genes related to ethylene signal transduction during papaya ripening were systematically analyzed.One key gene in ethylene signal transduction,CpEBF1,was used as the bait protein to screen the yeast two-hybrid cDNA library.Interacting proteins were found and BiFC and GST-pull down assays were used to further verify their interaction with CpEBF1.Transient expression analysis further investigated the role of their interactions in regulating genes encoding cell wall degrading enzymes.The main results were as follows:1.Improper treatment of 1-MCP leading to fruit“rubbery”ripening disorder.significantly inhibited the physiological and biochemical changes associated with fruit softening.Inappropriate 1-MCP treatment(400 nL·L-1 1-MCP treatment for 16 h)significantly inhibited the ethylene release and respiration rate of papaya,and the pulp could not completely soften,showing a“yellow peel,hard pulp”and“rubbery”ripening disorder.Comparatively,the appropriate 1-MCP treatment(400 nL·L-1 1-MCP treatment for 1 h)could delay fruit ripening,but no softening disorder appeared.Inappropriate 1-MCP treatment inhibited the normal degradation of cell wall structure,promoted the increase of lignin content,and inhibited the expression of cell wall softening-related genes including CpPG1/2,CpEXP1/2,and CpPMEs,which could be an important cause of ripening and softening disorders.2.The interaction between CpEBF1 and CpMADS1/3 regulated the disorder of papaya ripening induced by 1-MCP.Inappropriate 1-MCP treatment significantly inhibited the expression of CpEBF1,whereas in the normal ripening fruit CpEBF1 was highly expressed at the initiation of ripening.The CpMADS1/3 and CpEIL1 proteins were screened out in the yeast two-hybrid cDNA library?using improper 1-MCP-treated sample?using CpEBF1 as bait.Y2H,BiFC,and GST-pull down technologies verified the interactions among them.Subcellular localization showed that they were all located in the nucleus.Transient expression showed that the interaction between CpEBF1 and CpMADS1/3 regulated fruit softening by activating the promoter activity of CpPG1/2 and CpEXP1/2 genes.3.Low temperature stress caused fruit ripening disorder and affected ripening-related physiological and biochemical changes.Papaya showed symptoms of chilling injury and ripening disorder which could not turn yellow and soften when transferred to room temperature after 7? for 25 days.The cell wall became thinner with lighter staining,and the nuclear membrane and the plasma membrane were damaged or even disintegrated.The structure of chloroplast disappeared,and the content of cell wall lignin increased,due to the increase of gene expression and related enzyme activity for lignin synthesis.Low temperature stress aggravated cell membrane damage and disrupted the stability and integrity of the membrane structure resulting in abnormal membrane lipid metabolism.The malondialdehyde?MDA?,cell membrane permeability and lipoxygenase?LOX?activity were increased.Low temperature stress increased the relative content of saturated fatty acids such as stearic acid?C18:0?,palmitic acid?C16:0?,but decreased the relative amounts of unsaturated fatty acids?C18:3?and linoleic acid?C18:2?content.In addition,low temperature stress caused an imbalance of reactive oxygen and endogenous hormone metabolism.4.CpEBF1 interacted with CpJAZ3 to control the ripening disorder of papaya caused by low temperature stress.Low temperature stress strongly induced the expression of CpEBF1,but the expression of CpEBF1 was inhibited after fruit ripening at room temperature.Using CpEBF1 as the bait protein in the yeast two-hybrid cDNA library?using lowtemperature-stressed sample?to screen interacting proteins.CpJAZ3 was identified and BiFC and GST-pull down verified their interactions.Low temperature stress significantly induced the expression of CpJAZ3,and subcellular localization showed its localization in the nucleus.In addition,Y2H,BiFC,and GST-pull down studies showed that CpJAZ3 also interacted with CpERF9,and CpERF9 expression was also significantly induced by low temperature stress,but its expression decreased significantly after the fruit was transferred to room temperature.Transient expression analysis showed that CpJAZ3 interacted with CpEBF1 and CpERF9 to inhibit the promoter activity of cell wall degrading genes CpPG2 and CpPME1 and affected the transcription of these softening-related genes.5.Construction of regulatory network of papaya ripening disorder caused by 1-MCP and low temperture stress mediated by CpEBF1 and its interacting proteins.Inappropriate 1-MCP treatment significantly inhibited ethylene production and the expression of most genes related to ethylene signal transduction pathway.The effect of ethylene response on fruit ripeing and softening was obviously reduced and the expression of CpEBF1 and CpMADS1/3 were almost completely inhibited.Then,the interaction between CpMADS1/3 and CpEBF1 was decreased,which prevented the activation of fruit softening-related genes and the changes of corresponding enzyme activity.Low temperature stress decreased the sensitivity of CpEBF1 to ethylene,inhibiting ethylene signal,and affecting normal fruit ripening.In addition,low temperature stress repressed the production of JA and induced the expression of CpJAZ3.The interaction between CpJAZ3 and CpEBF1,CpERF9 were increased,which promoted the suppression of fruit softening-related genes. |
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