Auxin-activated MdARF5 Induces The Expression Of Ethylene Biosynthetic Genes To Initiate Apple(Malus Domestica)fruit Ripening

Apple(Malus domestica)is the most cultivated fruit tree in the world,its delicious taste and fantasy peel color makes it popular in people.But the maturation period of different apple is close,thus to maintain the constant supplying,apple fruit was often stored at specific conditions to prevent its ripening before sale.Moreover,apple is a typical climacteric fruit whose full ripening relies on the participation of the gaseous plant hormone ethylene.Thus,the theory of which how ethylene produce during fruit development and ripening is crucial for artificially regulating fruit ripening period and enhancing the fruit storability.Ethylene production during fruit ripening is a complete physiological process,and is influenced by many aspects,such as transcription factors,plant hormones.The auxin is a basic hormone controlling varied physiological process in higher plant,recent reports revealed that auxin is able to influence the ethylene production during fruit ripening,but how auxin signaling regulates ethylene production still needs further study.In our study,a synthetic stable form of auxin,naphthaleneacetic acid(NAA),was used to treat the ‘Golden Delicious' apple fruit.We found that NAA induced ethylene production and initiated fruit ripening process even when the fruit is not otherwise of ripening.An auxin response factor,Md ARF5,which is stimulated by auxin,could induce the expression of Md ACS3 a,Md ACS1 and Md ACO1 through GUS activity assay,and Chromatin immunoprecipitation(Ch IP)-PCR analysis further confirmed that Md ARF5 bound to their promoters.The representative results are as follow:1.Apple fruit was harvested at commercial harvest day(145 days after harvest,DAFB),and treated with NAA,then stored at room temperature 20 d.Results showed that NAA treatment promoted the ethylene production of fruit and accelerated apple fruit ripening.2.To explore when auxin can promote ethylene production in apple.Apple fruit was harvested every 30 d since 55 DAFB,treated with NAA.Results turned out that auxin induced fruit ripening and ethylene production,as well as the expression of Md ACS3 a,Md ACS1 and Md ACO1,at 115 DAFB on which apple fruit is not otherwise of ripening naturally.3.Apple fruit samples at 5 DAH(days after harvest)which was treated with or without NAA at 115 DAFB was used for transcriptome analysis.The RNA-Seq data revealed five differentially expressed Md ARF genes.Quantitative real time(q RT)-PCR assay showed although all Md ARF genes were stimulated by NAA treatment,but only Md ARF5 shared the same expression pattern with ethylene manufacture tendency.4.To verify the role of Md ARF5 in apple fruit ripening,Md ARF5 was silenced in apple fruit using Agrobacterium transfection system.We observed that suppressing of Md ARF5 reduced the auxin-induced ethylene production,as well as the expression of ethylene biosynthetic genes.By contrast,Overexpression of Md ARF5 showed opposite results with that of its silencing fruit.5.To analysis the regulation of Md ARF5 to ethylene biosynthetic genes,Ch IP-PCR,GUS activity assay and EMSA were performed.Results revealed that Md ARF5 bound to promoters of ethylene biosynthetic genes,such as Md ACS3 a,Md ACS1 and Md ACO1,and positively regulated their expression.6.From our RNA-seq data,we obtained a differentially expressed gene,Md ERF2,which was proved that its expression was significantly induced in NAA treated fruit.To clarify the regulation of Md ARF5 to ethylene signaling,we used Ch IP-PCR,GUS activity assay and other experiments to reveal that Md ARF5 bound to promoter of ethylene response factor,Md ERF2,and stimulated its expression.7.Since both Md ARF5 and Md ERF2 regulated Md ACS3 a and Md ACS1 expression,we used GUS activity assay to investigate the regulation of ethylene production when they both existed.GUS activation assay represented that Md ARF5 together with Md ERF2 enhanced the activation of Md ACS3 a promoter;however,Md ERF2 suppressed the Md ARF5 activated Md ACS1 promoter activation.8.The apple fruit with NAA treatment in 95 DAFB harvested samples showed auxin induced the ethylene production only if Md ACS3 a expression initiated.The DNA methylation detection to its promoter indicated that Md ACS3 a expression is influenced by its promoter DNA methylation level,which was not affected by auxin.Taken together,our study revealed three paths for auxin-induced ethylene biosynthesis in apple fruit: after demethylation of the Md ACS3 a promoter,Md ACS3 a expression is induced.Subsequently,auxin-activated Md ARF5 promotes the expression of Md ACS3 a directly and indirectly through Md ERF2.Md ARF5 also promotes the expression of Md ACS1 and Md ACO1,and once Md ACS1 expression is initiated,Md ERF2 acts as a transcriptional repressor of Md ACS1,thereby modulating ethylene production.