The Molecular Mechanism Underlying The Synergistion Between Peach ERFs And PpIAA1 In Controlling Fruit Ripening And Softening

Based on the characteristics of fruit firmness and textural changes during ripening,peach cultivars can be classified into three groups: melting(M),non-melting(NM),and stony hard(SH)flesh types.In the melting flesh peach,rapid softening occurs after harvest,resulting in a short shelf life.By contrast,stony hard peaches barely soften on the tree or after harvest,even though the fruits change color normally,contain high soluble solids,and have good flavor.This study was to elucidate the molecular mechanism by which auxin and ethylene synergistically regulate the ripening of peach fruit.MF and SH type peach fruit was used as samples to screening AUX/IAA and ERFs genes which showd a huge differences in gene expression between the two peach flesh types.Then Y1 H,EMSA,DLR,transient expression in peach fruit,Y2 H,BiFC and pull-down was used to study how AUX/IAA and ERFs regulated the expression of peach fruit ripening related genes,such as PG,ACS,ACO,NCED genes;as well as the relationship between AUX/IAA and ERFs.In order to further clarify the role of auxin and ethylene in the ripening process of peach fruit,and lay a theoretical foundation for the cultivation of new peach varieties with long shelf life and storage.1.Identification of ERF genes involved in ethylene signal transduction in peach.Through BLAST analysis of tomato(Solanum lycopersicum)protein sequences against the peach genome database and transcriptome analysis of all APETALA2/Ethylene Responsive Factor(AP2/ERF)genes in peach,we identified 15 AP2/ERF genes with significant different expression levels in CN16(stony hard)peaches and CN13(melting flesh)peaches during fruit maturation.Through quantitative RT-PCR expression analysis.Seven ERF genes were further identified.The transcript levels of Prupe.1G037700(PpERF1),Prupe.2G289500(PpERF7),Prupe.3G240000(PpERF6),Prupe.5G061800(PpERF5),Prupe.1G390800(PpERF4),Prupe.7G194400(PpERF3),Prupe.5G090800(PpERF2)were significantly different with that in CN16.2.PpERF2,PpERF3,PpERF4 regulated the expression of fruit ripening related gene.1)we analyzed the transcript profiles of PpNCED genes using transcriptome data and verified the results by qRT-PCR.ABA biosynthesis gene and cell wall degredation gene which shared a similar expression profile as candidate ERFs gene was identified.Furthermore,1-MCP,NAA or ethylene treatment could dramatically affect the expression of PpNCED2,PpNCED3 and PpPG1.Online analysis found ERF-binding motifs(CCGAC 、 A/GCCGAC 、 AA/TTTCAAA)in PpNCED2,PpNCED3 and PpPG1 promoter,and auxin response element(CCGACA,TGTCTC).PpERF2 was found to directly bind to the PpNCED2,PpNCED3 and PpPG1 promotors through CCGAC motif as demonstrated by yeast one-hybrid(Y1H)and EMSA assays;PpERF3 might bind to the promoters of PpNCED2,PpNCED3;PpERF4 could directly bind to PpNCED2,PpNCED3,PpPG1 and PpACO1 promoter via CCGAC,AGCCGCCA motif.Transient expression analysis suggested that PpERF2 was a repressor and could repress the promoter activities of the PpNCED2,PpNCED3 and PpPG1 genes;PpERF3 increased the expression of PpNCED2,PpNCED3;PpERF4 was a transactivitor and could increased the transcripton of PpNCED2,PpNCED3,PpPG1 and PpACO1.3.PpIAA1 regulates the ripening and softening of peach fruitIn total,22 AUX/IAA and 21 ARF genes have been identified in the peach genome.Based on our previous transcriptome data,these 21 ARF genes exhibited similar expression during fruit ripening in CN13 and CN16.Additionally,the treatment of CN13 with 1-MCP or of CN16 with ethylene and NAA did not result in obvious changes in the expression levels of the 21 ARF genes.Many of the 22 AUX/IAA genes were expressed at dramatically higher levels in CN13 than in CN16 at S4 III stage,including ppa010303,which showed a parallel expression pattern to the ethylene biosynthesis gene PpACS1.Using qRT-PCR,we further confirmed that the expression level of ppa010303(PpIAA1)in CN13 was significantly higher than in that of CN16.Y1 H and EMAS results indicated that PpIAA1(ppa010303)could directly bound to the PpNCED2,PpNCED3,PpPG1,and PpACS1 promoters via the CCGACA element in the PpNCED2 and PpPG1 promoters,the TGTCTC element in the PpNCED3 promoter,and the TGTG?TGTG element in the PpACS1 promoter.Transient expression assay showed that the promoter activity of PpNCED2,PpNCED3,PpPG1,and PpACS1 could be increased by PpIAA1.PpIAA1 and PpERF4 could bind together as demonstrated by Y2 H,BiFC and pull-down,and the binding of PpIAA1 between PpERF4 further enhanced the transcription of PpNCED2,PpNCED3,and PpPG1 than each transcription factor separately.Compared with WT,the transgenic lines did not have lateral branch and the lateral roots was reduced.PpIAA1 overexpression in tomato accelerated fruit ripening and shortened the fruit shelf life.Collectively,these results suggested: 1)Ethylene regulates the synthesis of abscisic acid by regulating the expression of abscisic acid synthesis gene PpNCED via PpERFs.2)PpIAA1 enhances the transcription of PpACS1,PpNCED2,PpNCED3 and PpPG1 by binding to its promoter;Moreover,3)PpIAA1 further enhances the transcription of PpNCED2,PpNCED3 and PpPG1 by interacting with PpERF4,and 4)PpERF4 can enhance the transcription of PpIAA1 by binding to its promoter.Auxin and ethylene control fruit ripening and softening in peaches through these four mechanisms underlying the cooperation between PpIAA1 and PpERF4.