| ‘Nanguo’ pears(Pyrus ussuriensis Maxim)are a specialty in the Liaoning province of China.They have the characteristics of delicate flesh,rich flavor,and rich nutrition,and is deeply loved by people.They are normally harvested in the first half of September.The harvested pears are not tolerated for storage,and after being stored at room temperature(20 ± 1 °C)for approximately half month,ripening and senescence,browning of the fruit core,and even rot will occur,reducing its commercial value.Refrigeration is a physical regulation technology that can effectively reduce the physiological metabolism level of fruits and delay the process of ripening and senescence of the fruit;therefore,it is widely used for the storage and preservation of fruits.Refrigeration can extend the storage period of ‘Nanguo’ pears to 6 months,but long-term refrigerated ‘Nanguo’ pears will appear pericarp browning during shelf life at room temperature,which seriously affects the quality and commercial value of ‘Nanguo’ pears.Therefore,it is of great theoretical and practical significance to study the mechanism of pericarp browning in refrigerated ‘Nanguo’ pears,find the molecular mechanism that regulates pericarp browning in refrigerated ‘Nanguo’ pears,and explore the regulation technology for alleviating pericarp browning in refrigerated ‘Nanguo’ pears.In this paper,‘Nanguo’ pears were used as the test material.By analyzing the relationship between pericarp browning and membrane lipid metabolism during refrigeration and subsequent shelf life at room temperature,finding the key genes that participate in membrane lipid metabolism,and analyzing the promoters of key genes and its cis-acting elements,screening transcriptional factors that may regulate key genes through yeast one-hybrid and electrophoresis mobility-shift assay(EMSA)tests,using GUS activity,dual luciferase,yeast two-hybrid and two bimolecular fluorescence complementary experiments to explore the transcriptional regulation mode of transcription factors on key genes.Based on the above-mentioned transcriptional regulation mechanism,the alleviation effect of control technology on the pericarp browning of refrigerated ‘Nanguo’ pears and its mechanism were studied.The results of this study will provide a reference for the research on the mechanism of chilling damage of other post-harvested vegetables and fruits,and provide a theoretical basis for exploring the control techniques for reducing the pericarp browning of refrigerated ‘Nanguo’ pears.The main results of this study are as follows:Changes in the appearance,cell structure,membrane lipid composition,and membrane lipid metabolism-related enzyme activities of ‘Nanguo’ pears,which were refrigerated for 60 d(short-term refrigeration)and 120 d(long-term refrigeration),were different.The results showed that long-term refrigerated fruit exhibited the symptoms of chilling injury of pericarp browning,severe cell membrane damage and organelle damage during shelf life at room temperature.In addition,the relative leakage rate and malondialdehyde(MDA)content in long-term refrigerated pears increased with the extension of shelf life,and were significantly higher than those in short-term refrigerated samples.Furthermore,the membrane lipid content in long-term refrigerated ‘Nanguo’ pears was reduced,among of which,phosphatidylcholine(PC),phosphatidic acid(PA),phosphatidylethanolamine(PE),lysophosphatidylcholine(LPC),and monogalactosyldiacylglycerol(MGDG)and digalactosyldiacylglycerol(DGDG)have been found to respond significantly to long-term low temperature stress.The relative content of saturated fatty acids increased,the relative content of unsaturated fatty acids decreased,and fatty acid unsaturation and unsaturated index decreased significantly in long-term refrigerated ‘Nanguo’ pears.Moreover,the activities of phospholipase D(PLD),lipase,and lipoxygenase(LOX)increased,and the expression of Pu PLDβ1 increased significantly,indicating that long-term refrigeration can induce the increases of PLD,lipase,and LOX activities,thereby changing the membrane lipid composition,destructing the cell membrane structure,reducing its fluidity and increasing its permeability,which lead to the contact between phenol enzyme with phenolic substrates,and result in pericarp browning,during this process,Pu PLDβ1 is the key gene that causes membrane lipid peroxidation and pericarp browning in refrigerated ‘Nanguo’ pears.Based on the occurrence of pericarp browning in refrigerated ‘Nanguo’ pears and the response of key genes in the membrane lipid metabolism pathway,Pu PLDβ1 was identified as a key gene involved in this process.Therefore,the Pu PLDβ1promoter sequence was cloned and analyzed.The analysis revealed that there were multiple elements related to abiotic stress and hormone response in the promoter sequence,as well as multiple transcription factor binding sites.According to the existing transcriptome data of refrigerated ‘Nanguo’ pears in our research group,two MYB transcription factors,Pu MYB21 and Pu MYB54,which can bind to the Pu PLDβ1 promoter were screened by yeast one-hybrid;EMSA test found that Pu MYB21 and Pu MYB54 specifically bind to the TAACTG site in the Pu PLDβ1 promoter;GUS and dual luciferase tests found that both Pu MYB21 and Pu MYB54 can activate the transcription of the reporter genes which were on the downstream of Pu PLDβ1 promoter,and the activation ability is stronger when the two coexist;overexpression of Pu MYB21 and Pu MYB54 in Nanguo pear fruit and callus resulted in increased transcription level of Pu PLDβ1,and interference of Pu MYB21 and Pu MYB54 expression decreased Pu PLDβ1 transcription level;yeast two-hybrid,GST-pull down,and bimolecular fluorescence complementary test showed that Pu MYB21 interacted with Pu MYB54;Real-time PCR showed that Pu MYB21 and Pu MYB54 were induced by long-term low temperature stress;amino acid sequence alignment and phylogenetic tree analysis found that Pu MYB21 and Pu MYB54 are R2R3 type MYB transcription factors,and Pu MYB21 was closed to white pear Pbr MYB21,while Pu MYB54 was closed to apple Md MYB54;subcellular localization results showed that these two MYB transcription factors were localized in the nucleus.The above results confirmed that Pu MYB21 and Pu MYB54 were involved in the degradation of membrane lipids under long-term low temperature stress by regulating the transcription of Pu PLDβ1 by targeting the TAACTG element on the Pu PLDβ1 promoter.Based on the transcriptional regulation mechanism of pericarp browning in refrigerated ‘Nanguo’ pears,combined with the antioxidant properties of MT,the regulation effect of MT treatment on pericarp browning in refrigerated ‘Nanguo’ pears was further explored.The results showed that the occurrence time of pericarp browning was delayed when pears were treated with MT after refrigerated for 120 d,the browning incidence and browning index were significantly reduced,the pericarp cell membrane was intact and the organelles were intact.In addition,after MT treatment,the relative leakage rate and MDA content decreased,proline synthase activity increased,expression level increased,degradation enzyme activity decreased,expression level decreased,and proline content increased.MT treatment inhibited the degradation of PC and PE,and increased the fatty acid unsaturation and unsaturated index.Furthermore,antioxidant enzyme activity increased,membrane lipid metabolism-related enzyme activity decreased,and the transcription levels of Pu MYB21,Pu MYB54,and Pu PLDβ1 genes were significantly reduced in the treatment group.The results showed that MT treatment might stimulate the antioxidant system of refrigerated ‘Nanguo’ pears,reduce the transcription of Pu MYB21 and Pu MYB54,thereby inhibited the expression of Pu PLDβ1,reduced the occurrence of long-term low temperature-induced membrane lipid peroxidation,and further enhanced the resistance ability to the long-term low temperature stress. 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