| Broccoli(Brassica oleracea L.var.italica)is rich in a variety of vitamins,antioxidants,glucosinolates and other bioactive ingredients,which is deeply loved by consumers.Postharvest broccoli undergoes vigorous metabolism and is not resistant to preservation,is prone to yellowing at room temperature,accompanied by the loss of nutrients,in severe cases,it will mildew and produce peculiar smell,which affects its commercial quality and value.Thus,studying the yellowing mechanism of broccoli and exploring regulatory techniques that can alleviate its quality deterioration have considerable theoretical significance and practical value.Chlorophyll is a major pigment substance in broccoli,and its degradation is the main factor of broccoli yellowing.Ethylene is recognized as a plant hormone that promotes post-ripening and senescence in plants.Previous studies in our laboratory have confirmed that ethylene can accelerate broccoli from green to yellow by promoting chlorophyll degradation.Jasmonic acid(JA),another important phytohormone in plants,also plays a crucial regulatory role in post-ripening senescence,and our preliminary study found that JA accumulates in broccoli as it changes from green to yellow.On this basis,firstly,methyl jasmonate(Me JA)and sodium diethyldithiocarbamate(DIECA),the inhibitor of JA synthesis,were used in this paper to analyze the effect of JA on broccoli yellowing by observing the changes of flower balls color and anatomical structure of flower buds during the shelf life,monitoring indexes such as chlorophyll content and chlorophyll fluorescence as well as the changes of JA content.Moreover,the key factors affecting JA synthesis during broccoli yellowing were studied by measuring the changes of JA content and key enzymes activity of JA synthesis in broccoli under different treatments,as well as the expression changes of differentially expressed JA synthesis genes screened based on transcriptome sequencing results.Focusing on the key gene of JA synthesis,molecular biology techniques were used to study the transcriptional regulation mechanism of transcription factors on this gene,so as to reveal the molecular mechanism of transcription factors mediating postharvest broccoli yellowing through regulation of JA synthesis.Furthermore,on the basis of the above mechanism research,the control technology to alleviate the yellowing of postharvest broccoli was further explored.Findings:(1)Both Me JA and DIECA treatments had certain effects on yellowing of broccoli.After Me JA treatment,the total chlorophyll,chlorophyll a and chlorophyll b contents decreased obviously in broccoli,the activities of chlorophyll degrading enzymes PPH and PAO increased drastically,and the expression of chlorophyll degrading enzyme genes BoPPH and BoPAO increased significantly.However,DIECA treatment obviously inhibited the decline of chlorophyll level and the up-regulation of chlorophyll degrading enzyme activities and related gene expressions.The content of JA in broccoli treated with Me JA increased significantly,the activities of JA synthase AOC and OPR increased,the expression of BoOPR1 was significantly up-regulated,while DIECA treatment distinctly inhibited the changes of JA synthesis related indicators.The correlation analysis results demonstrated that the change of JA content was significantly positively correlated with the change of flower bulb yellowing index,and negatively correlated with its total chlorophyll content.The results of orthogonal partial least squares discriminant analysis(OPLS-DA)showed that BoOPR1 gene expression was significantly positively correlated with the change of JA content.Based on the above analysis,it can be seen that JA accumulation is closely related to the yellowing in postharvest broccoli,BoOPR1 is the key gene for JA synthesis,and its up-regulated expression promotes the synthesis of JA,which may be an important factor in the yellowing of broccoli.(2)The BoOPR1 gene fragment was ligated to the p RI101 overexpression vector and the p TRV vector to obtain the overexpressed recombinant plasmid and silenced recombinant plasmid,and BoOPR1 was overexpressed and silenced by agrobacterium-mediated technology,respectively.The expression level of BoOPR1 gene in broccoli after infection was verified,and after successful transformation,compared with the control,the overexpressed BoOPR1 samples exhibited obvious yellowing,and the electron microscopic observation showed severe cell shrinkage,abnormal stomatal morphology,massive disintegration of chloroplasts.Conversely,the silenced BoOPR1 samples showed stay-green phenotype,the cell surface was smooth,complete and orderly arranged,and the chloroplast remained in good shape.The JA content of samples overexpressed with BoOPR1 gene increased significantly,and the total chlorophyll content decreased obviously,while silencing BoOPR1 gene reduced the JA content,and the degradation of total chlorophyll was also inhibited.The above results further verify the important role of BoOPR1 gene in JA synthesis and broccoli yellowing.(3)In view of previous transcriptome sequencing results,the BoOPR1 promoter sequence was cloned,and its cis-acting element information was analyzed.Yeast one-hybrid assay showed that BoMYB315 and BoBEE3 could bind to the BoOPR1 promoter.Electrophoresis mobility-shift assay showed that BoMYB315 and BoBEE3 selectivity bound to TAACCA and CACATG motifs on the promoter of BoOPR1.GUS reporter activity analysis and dual-luciferase assay demonstrated that BoMYB315 and BoBEE3 had transcriptional activation activity,both of which could activate the expression of BoOPR1 gene,and the regulatory ability was stronger when they coexisted.The results of RT-PCR analysis showed that BoMYB315,BoBEE3 and BoOPR1 genes were remarkably up-regulated during the yellowing process of broccoli.The expression of BoOPR1 was significantly up-regulated when BoMYB315 and BoBEE3 were transient overexpressed in broccoli and callus,while the expression of BoOPR1 was obviously down-regulated by silencing BoMYB315 and BoBEE3.The regulatory effects of transcription factors BoMYB315 and BoBEE3 on BoOPR1 were verified.Yeast two-hybrid and bimolecular fluorescence complementation experiments showed that there was interaction between BoMYB315 and BoBEE3.In addition,BoMYB315 and BoBEE3 are nuclear localization proteins,which separately belong to the R2R3 type MYB family and b HLH transcription factor family.These results indicated that transcription factors BoMYB315 and BoBEE3 were involved in JA synthesis through coordinately positive regulation of the expression of BoOPR1,thereby affecting the yellowing of broccoli.(4)The yellowing symptoms of broccoli treated with 2,4-epibrassinolide(EBR)were visibly reduced during the shelf life.The total chlorophyll and chlorophyll a level in treated samples was remarkedly higher than that in the control,and the expressions of BoPPH,BoNYC1 and BoSGR1,key genes for chlorophyll degradation,were distinctly inhibited.The JA content in EBR-treated broccoli was markedly lower than that in the control,the activity of JA synthetase OPR was decreased,and the up-regulated expressions of BoOPR1,the key gene for JA synthesis and transcription factor genes BoMYB315 and BoBEE3,were also obviously inhibited.The above results further verify the mechanism research results in this paper,and also provide new insights for improving the postharvest treatment technology of broccoli in production and extending its shelf life. |
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