Molecular Mechanism Of CsTT8 Involved In Regulating Fruit Quality In Blood Orange (Citrus Sinensis)

Blood orange is a cultivated of citrus sinensis,which fruit are rich in anthocyanins.A b HLH transcription factor NOEMI is involved in the accumulation of organic acid in fruits and anthocyanins in seed coat.A b HLH-type transcription factor,containing basic helix-loop-helix,have different physiological functions in plants,which are able to regulate growth and respond to kinds of stress.In most reports,b HLH transcription factor were involved into anthocyanin biosynthesis and vacuolar acidification in model plants.In fruit trees,the b HLH-type transcription factors are still unclear about threir functions.In this study,the fruit qualities of blood orange were measured in comparison with two varieties.The full-length of coding sequence of a b HLH transcription factor from juice vesicles in blood orange was obtained.So that it was designated by CsTT8 in blood orange.Besides,an alternative splicing transcript of b HLHΔ15 were found in different varieties.Using quantitative measurement of differential gene expression,subcellular localization,biological function identification,yeast two-hybrid,yeast one-hybrid,dual luciferase reporter system were performed to study CsTT8 and CsTT8Δ15.The esults as follows:(1)Comparative analysis of fruit quality between two cultivars of blood orangeThere was no difference in external quality between two varieties.Therefor the total soluble solids in the fruits between two varieties were 10.5% and BO3 showed the characteristics of low citrate and low anthocyanin accumulation,and BO9 showed the characteristics of high citate and high anthocyanin accumulation.(2)CsTT8 cloning and differential gene expression analysisA transcription factor was cloned in blood orange with a full length of 2085 bp,which encoding 695 amino acid residues with b HLH-MYC domain at N-terminus and helix-loop-helix domain at C-terminus,named as CsTT8.At addition,different alternative splicing transcripts of CsTT8 were found in blood orange.The genes involving into anthocyanin biosynthesis pathway had significantly different between two varieties of blood orange.In the BO3 with lower anthocyanin accumulation,the relative expression levels of CsTT8,Cs RUBY,Cs CHS,Cs F3’H,and Cs GST were significantly lower than those of BO9.Besides,the up-regulated expression of above-mentioned genes were positively correlated with anthocyanin accumulations in juice vesicles(r values >0.7,p ≤0.05).However,there was no significant different in the transcription level of Cs WD40 between two varieties.(3)Research on the interaction between CsTT8 and other transcription factorsYeast two-hybrid experiments showed that CsTT8,Cs RUBY,and Cs WD40 interacted with each other,but CsTT8Δ15 could not interact with Cs RUBY.Recombination protein which contain difference domain interact with Cs RUBY and Cs WD40,illuminate that b HLH-MYC domain at the N-terminus of CsTT8 was able to interact with Cs RUBY,and helix-loop-helix domain at the C-terminus of CsTT8 was also interact with Cs WD40.(4)Research on subcellular localization and CsTT8 gene functionThe results of subcellular localization showed that only CsTT8 and Cs RUBY were able to enter into nucleus.In the transient expression analysis,only the combination of CsTT8,Cs RUBY and CsTT8,Cs RUBY,Cs WD40 were able to upregulate anthocyanin accumulations in blood orange.(5)Research on CsTT8 transcription activationYeast one-hybrid and dual-luciferase experiments show that combination of CsTT8 and Cs RUBY can activate the expression of reporter of F3’H,and Cs WD40 can strengthen the transcriptional activation activity.Besides,combination of CsTT8 from blood orange and Cs PH4 from navel orange can activate expression of PH8.