Research On The Mechanism Of Anthocyanin Accumulation Responding To Light And Low Temperature In Fruits Of Blood Orange And Purple Pummelo

Anthocyanins,which belong to flavonoids,have been widely reported to play important roles in plant adversities resistance and human health.Citrus is the most productive fruit in the world,and a few citrus species can accumulate anthocyanins in a species or tissue specific manner.In wild and cultivated citrus sepecies,Ruby1 is a R2R3-MYB transcription factor which had been functionally identified and confirmed to be positive regulator for anthocyanin accumulation.Notably,environmental factors,such as light and temperature,can affect the synthesis of citrus anthocyanins by regulating the expression of Ruby1.Previous studies have found that low temperature and light can promote the accumulation of anthocyanins in citrus fruit.However,the mechanism behind light and low temperature induced anthocyanin accumulation in citrus fruits remain poorly understood.Blood orange and purple pummelo are two unusual citrus accessions for anthocyanin accumulation in their mature fruits.Blood orange accumulated anthocyanin in both its fruit peel and flesh whereas purple pummelo accumulated anthocyanin only in its fruit peel.Based on the previous study of citrus anthocyanin biosynthesis in our laboratory,blood orange and purple pummelo were used as materials to further explore the regulatory mechnism of light and low temperature induced anthocyanin synthesis in citrus fruits.The main results are as follows:1.Light can induce anthocyanin accumulation in both blood orange and purple pummelo fruit peel.Through the bagging experiments,we found that the accumulation of anthocyanins in fruit peel of blood orange and purple pummelo can be induced by light.The analyses of promoter elements and the detection of promotor activity indicated that Ruby1 promotor in blood orange(Cs Ruby1)can be induced by light and low temperature,whereas Ruby1 promoter in purple pummelo(CgRuby1)can only be induced by light.2.Localization of key light-responsive element in CgRuby1 promoter.We fused the full length and three truncated fragments of the CgRuby1 promoter to the GUS reporter gene,and these constructs were transformed into Arabidopsis.We detected the GUS activity of T3 transgenic lines under different light conditions.The result indicated that light responsive elements distributing in the CgRuby1 promotor from 0 bp to-234 bp is a key region which contributed to light responsiveness.3.A proposed model for the induction of anthocyanin synthesis by light and low temperature stimuli.By analyzing the Ruby1 promotor sequence in different citrus species,we found that G-box element in the Ruby1 promotor involved in light responsiveness were conserved in different citrus.In addition,the results of electrophoretic mobility shift(EMSA)assay and dual luciferase assay showed that HY5 can bind to the G-box(CACGTC)elements,and positively regulate the expression of Ruby1,thereby increase the accumulation of anthocyanins.4.Exploring of transcription factors responding to low-temperature induced anthocyanin biosynthesis in blood orange.Three bHLH transcription factors(bHLH1、bHLH2 、 bHLH3)responding to low temperature were identified by analysing the trascriptome data from citrus fruits.The expression of these three transcription factors in fruits were induced by low temperature.The exact mechanism deserved further study.