Identification And Transcriptional Regulation Of ACL Genes In Citrus Fruits

Organic acid is one of the most important contributors for fruit flavor.In citrus fruits,the predominant organic acid is citric acid.In this study,Ponkan fruit(Citrus reticulate Blanco cv.Ponkan)and Miyagawa wase fruit(Citrus unshiu Marc.cv.Miyagawa wase)were used as materials,and the roles of the Cit ACL during citrus fruit development were studied.Then one transcription factor,Cit ERF6,was observed to play important roles in transcriptional regulation on Cit ACLA3.The main results are as follows:1.Isolation and characterization of Cit ACL genes.Three Cit ACL genes,Cit ACLA2,Cit ACLA3 and Cit ACLB were isolated from the Citrus Genome Database.Sub-cellular localization showed that Cit ACLA2 was mainly targeted to both nucleus and cytoplasm,while Cit ACLA3 and Cit ACLB were mostly located at the cytoplasm.2.Cit ACLA3 regulated citric acid degradation in citrus fruit.The relationship between Cit ACL and citric acid was investigated,using the citrus varieties ‘Ponkan’ fruits and ‘Miyagawa wase’ fruits.The expression profiles Cit ACLA3 was highly correlated to citrate degradation.These results indicated that Cit ACLA3 may be related to citric acid degradation.In addition,transient over-expression assay showed that Cit ACLA3 could reduce citric acid contents in citrus leaves,supporting the potential role of Cit ACLA3 in citric acid degradation.3.Cit ACLA3 was transcriptional regulated by Cit ERF6.The transcriptional regulation of Cit ACLA3 was studied and a transcription factor,Cit ERF6,was found.Dual-luciferase assay indicated that Cit ERF6 acted as an activator,which significantly activated the promoter activity of the Cit ACLA3.Then EMSA showed that Cit ERF6 could bind to the specific sequence in Cit ACLA3 promoter.Taken together,these results suggested that Cit ERF6 may play a role in citrate degradation via directly binding and activating the promoter of Cit ACLA3.