Mechanism Analysis Of NAC Transcription Factors In Regulation Of Carotenoid Biosynthesis During Papaya Fruit Ripening

Papaya(Carica papaya L.)is a typical climacteric fruit and rich in nutritional compounds,such as carotenoids.Carotenoids are important nutrients for human health,however,humans and almost all animals do not synthesize carotenoids do nevo,and they are sourced only from diet.Carotenoids of flesh papaya fruit are closely related to fruit nutritional quality.Papaya can be classed into red-flesh and yellow-flesh according to the color of fruit pulp,and it has been reported that primarily lycopene in red-flesh,while,largely ?-carotenids and ?-cryptoxanthin in yellow-flesh.In addition,many more carotenoids were accumulated in red-flesh rather than yellow-flesh papaya during fruit ripening.Carotenoid biosynthetic genes,like PSY,PDS,ZDS,LCY-e,LCY-b,and CHY-b,have a strong impact on carotenoids accumulation.Therefore,expanding the knowledge of regulation mechanisms of carotenoid metabolism and carotenoids accumulation mechanisms of various cultivars during papaya fruit ripening do have important theoretical direction to improve fruit quality.NAC(NAM,ATAF1/2 and CUC2),a plant-specific transcription factor(TF),plays important roles in plant growth,development,stress responses and response to hormone signal.In addition,researches have shown NAC transcription factors were involved in carotenoid accumulation of tomato fruit.However,it is still unclear whether and how NACs involved in carotenoid biosynthesis during papaya fruit ripening.Therefore,in this study,we regarded two varieties ‘Suiyou-2' and ‘Suihuang' as objections,which exhibited different carotenoids accumulation.We analysis the mechanisms of NAC transcription factors involved in regulating of carotenoid biosynthesis during papaya fruit ripening.The mainly results are as follows.1.7,111 differentially expressed genes were detected as expressed during ‘Suiyou-2' and ‘Suihuang' papaya fruit ripening,and more of them are involved in fruit ripening rather than differentially expressed between ‘Suiyou-2' and ‘Suihuang'.These annotated differentially expressed genes were mainly grouped into 36 categories,including molecular function,biological process and cellular component.The most of differentially expressed transcription factors(TFs)is bHLH,followed are WRKY,MYB,NAC,and ERF.11 carotenoid biosynthetic genes were found to be differentially expressed among four comparisons.qRT-PCR assays were further performed to confirm expression patters of the 11 differentially expressed carotenoid biosynthetic genes during papaya ripening,the results are consistent with the results of RNA-seq,indicating that the data obtained by RNA-Seq are reliable.2.CpNAC1 involved in carotenoid biosynthesis through transcriptional activating the expression of CpPDS2/4,CpZDS,CpLCY-e,and CpCHY-b.qRT-PCR showed that CpNAC1 was up-regulated during fruit ripening,and the transcript levels of CpPDS2/4,CpZDS,CpLCY-e,and CpCHY-b were ripening-induced,correlated well with the accumulation contents of carotenoids during fruit ripening.In addition,electrophoretic mobility shift assay(EMSA)showed that CpNAC1 could directly bind to CpPDS2/4,CpZDS,CpLCY-e,and CpCHY-b promoters.Dual-luciferase reporter system(DLR)demonstrated CpNAC1 has transcriptional activation activity and transcriptional activated CpPDS2/4,CpZDS,CpLCY-e,and CpCHY-b promoters activity.The results indicated that CpNAC1 acts as a transcriptional activator,and regulates carotenoid biosynthesis through transcriptional activating the expression of carotenoid biosynthetic genes during fruit ripening.3.Papaya CpNAC2 and CpEIN3 a involved in carotenoid biosynthesis during fruit ripening,via activating the transcripts of CpPDS2/4,CpLCY-e,and CpCHY-b.qRT-PCR showed that CpNAC2 and CpEIN3 a were up-regulated during fruit ripening.EMSA demonstrated that CpNAC2 could directly bind to CpPDS2/4,CpZDS,CpLCY-e,and CpCHY-b promoters,while CpEIN3 a could directly bind to CpPDS4 and CpCHY-b promoters.Both Yeast two-hybrid(Y2H)and biomolecular fluorescence complementation(BiFC)assay demonstrated the interaction between CpNAC2 and CpEIN3 a.DLR assay indicated that both CpNAC2 and CpEIN3 a showed transcriptional activation activities,with CpNAC2 activating CpPDS2/4,CpLCY-e,and CpCHY-b promoters,while CpEIN3 a activating the promoters of CpPDS4 and CpCHY-b.More interestingly,the transcriptional activation abilities of reporters CpPDS2 and CpLCY-e were more pronounced when CpNAC2 and CpEIN3 a co-transformed.Collectively,our results suggest that CpNAC2 interacted with CpEIN3 a and,individually or cooperatively,activated the transcription of a subset of carotenoid biosynthetic genes CpPDS2/4,CpLCY-e,and CpCHY-b,involved in carotenoid biosynthesis during papaya fruit ripening.