The Regulation Mechanism Of Delayed Ripening And Senescence Of Green Ougan

Citrus is the representative non-climacteric fruit,the ripening mechanism and postharvest biology characteristics are completely different from the model plants such as Arabidopsis and rice,and also different from climacteric fruits?such as tomato and apple?.Recently,the researches about the ripening and senescence of citrus mainly focus on the descriptive study of this process through several parameters?such as skin color,sugar and acid content and postharvest rotting rate?while the mechanism study of this process is rare.The previous study in our lab had constructed the regulation network of postharvest citrus fruit senescence,and found that transcription factor and hormone may play important roles in the ripening and senescence process of citrus fruit,which lacked the experimental evidence.Therefore,in the present study,the difference of ripening and senescence phenotypes of Ougan?Citrus reticulata cv.Suavissima?,Green Ougan and the revertant of Green Ougan have been analyzed,and the transcriptional regulation mechanism of carotenoid and ABA metabolism have been parsed.To further verify the effect of exogenous hormone treatment on the postharvest citrus fruits,the effect of salicylic acid?SA?and brassinolide analogue,24-epibrassinolide?EBR?on Citrus unshiu has been analyzed.The main results are as follows:1.The Green Ougan was a delayed ripening and senescence bud mutants.The phenotypes analysis of the three materials indicated that the ripening and senescence processes?such as the color change and the decline of the organic acid content of the pulp?of Green Ougan were significantly delayed.After determination the chlorophyll and carotenoid of the flavedo,the results indicated that the decline of chlorophyll and carotenoids which were abundant in chloroplast?such as?,?-carotene and lutein?of Green Ougan were significantly slower than that of in Ougan.And the contents of the downstream products of?-carotene??-cryptoxanthin,zeaxanthin and 9-cis-violaxanthin?were significantly lower in Green Ougan than that of in Ougan.Moreover,ABA content of Green Ougan did not increase with the ripening process,and was significantly lower than that of in Ougan.The differences of chlorophyll,carotenoids and ABA content between Green Ougan and the revertant were similar to those of Green Ougan and Ougan.The above results indicated that the delayed ripening and senescence of Green Ougan were due to the postponed transformation of?,?-carotene to the downstream products which blocked the chloroplast transformation and the pigment synthesis of ripened fruit,and postponed synthesis of ABA which delayed the ripening initiation.2.CrMYB68 and CrNAC036 regulated the ripening and senescence of Green Ougan through repressing the genes involved in carotenoid and ABA metabolism.To parse the intrinsic mechanism of the difference of carotenoids and ABA content between Green Ougan and Ougan,the present study analyzed the expression of the genes involved in carotenoid and ABA metabolism and found that the postponed transformation of?,?-carotene and the synthesis of ABA were due to the lower expression of CrBCH2 and CrNCED5 in Green Ougan than that of in Ougan.After the analysis of the transcriptional data of the two materials,the results indicated that the expression of CrMYB68 and CrNAC036 was significantly negatively correlated with the expression of CrBCH2 and CrNCED5.The expression differences of these four genes were further confirmed in Green Ougan and the revertant.The Selected Amplification And Binding?SAAB?experiment indicated that CrMYB68 protein can specifically bind to ACCTAC,ACCAAC and AACAAC sequences.The promoter analysis of the CrBCH2 and CrNCED5 and the Electrophoretic Mobility Shift Assay?EMSA?indicated that CrMYB68 can specifically bind to the promoter of CrBCH2 and CrNCED5.The results of dual luciferase assay showed that the activity of CrBCH2 and CrNCED5 promoter were significantly inhibited by CrMYB68.The transient overexpression on the tobacco?Nicotiana benthamiana?leaves and EMSA signified that CrMYB68 can also bind to the promoter of NbBCH2 and NbNCED5 and inhibit the expression of these genes.The study of CrNAC036 found that it was able to bind to CrNCED5 promoter and inhibit its expression.After the analysis of the expression of genes induced by ABA,the study found that the expression of CrPSY involved in carotenoid biosynthesis,CrPectate Lyase and Cr Expansin A1/A8 involved in cell wall degradation was lower in Green Ougan than that of in Ougan.The above results showed that the delay of ripening and senescence of Green Ougan were attributed to the high expression of CrMYB68 and CrNAC036.On the one hand,the high expression of CrMYB68 can inhibit the CrBCH2 expression and then repressed transformation of?,?-carotene which affected the chloroplast degradation and the pigment synthesis of ripened fruit;on the another hand,the high expression of CrMYB68 and CrNAC036 can suppress the expression of the CrNCED5 and delayed the ABA synthesis which affected the initiation of ABA-depended ripening processes.These results have demonstrated that ABA played important roles in citrus ripening and senescence process,and the comprehensive analysis of the pre-experiment,regulatory network and the previous reports indicated that SA and brassinosteroid may act as the negative regulator on postharvest citrus ripening and senescence.3.Exogenous SA and EBR treatment can significantly improve postharvest storage performance of citrus fruits.In order to verify the effect of SA and EBR on the postharvest citrus fruit,the present study chose mandarin as material.In the present study,fruits were treated with 2 mmol L^-11 SA and 10?mol L^-11 EBR,respectively.The results showed that the SA treatment reduced the rotting rate after 50 d storage,and significantly delayed the decline of firmness during storage.The analysis of metabolites indicated that SA treatment can remarkably induce the accumulation of H₂O₂ and polymethoxylated flavone to enhance fruit disease resistance.Moreover,after the EBR treatment,the rotting rate of the EBR-treated fruit after 50 days storage was just 4%while that of the control is 18.7%.After the EBR treatment,fruit exhibited a rapid water loss process,which was similar to the phenotype of sweating treatment.In addition,the content of H₂O₂,serine,threonine,ornithine,proline and the expression of chitinase,phenylalanin ammonia lyase in EBR-treated fruit were higher than that of in the control fruits.The induction of these metabolites and genes enhanced the disease resistance of the fruit.