The Role And Mechanism Of PYLs Regulating Apricot Fruit Ripening

Apricot(Prunus armeniaca L.)is an important stone fruit of the Rosaceae family.During fruit ripening,the fruits occur many noticeable physiological changes such as softening,coloration,become fragrant,accompanied by the accumulation of carotenoids and other nutrients.Apricots belong to typical climacteric fruit and thus are ideal material for studying fruit ripening.Abscisic acid(ABA)is an important hormone for regulating fruit ripening.Its receptor gene PYLs are the core elements in ABA signal pathway.To date,it has been reported the role of PYLs in regulating ripening of non-climacteric fruit such as strawberry,however,it is still unknown whether PYLs in ABA signal pathway regulate climacteric fruit ripening.In this study,‘Danxing’ fruit were treated with exogenous ABA to reveal the effect of ABA on apricot fruit ripening.Based on weighted gene co-expression network analysis(WGCNA)of our previous transcriptome data,we obtained ABA receptor genes,genes related to ABA biosynthesis and signal transduction,and analyzed the response of these candidate genes to exogenous ABA during fruit ripening.Subsequently,the full length of candidate PYLs genes were cloned,and their putative function were analyzed by phylogenetic analysis and protein structure domain analysis.And then the function of one candidate genes Pa PYL9 was verified by using system.Furthermore,the response genes in transgenic tomato were screened by transcriptome sequencing,and ultimately the related regulatory network was proposed.These findings laid the foundation for unvein the molecular mechanisms underlying regulation fruit ripening of PYLs family members.The main findings are as follows:1.When ‘Danxing’ fruit before turning stage were treated with ABA,the color index of grape(CIRG)increased significantly,while the hardness of fruits decreased ramarkablely,accompanied by a marked increase of the total soluble solids(TSS)content and the sharp decreace of titratable acid(TA)content(p < 0.01).2.Based on gene co-expression network analysis(WGCNA),we obtained 12 candidate genes from apricot transcriptome data that may be involved in regulation of apricot fruit ripening underlying ABA signal pathway,including six ABA receptor genes(PYL9/RCAR1,U22307;PYL8/RCAR3,U13924;PYL4.1/RCAR10,U18739;PYL4.2/RCAR10,U22578;PYL1/RCAR12,U23298;PYL2/RCAR14,U16157),one NCED1(U19863)gene,three PP2Cs(PP2C11,U4803;PP2C33,CL539.2;PP2C27,U19425)genes,one Sn RK2(U3561)gene and one ABF2(U6847)gene.3.These candidate genes response to exogenous ABA was analyze by using q RT-PCR.The results showed that the expression levels of Pa NCED1,Pa PYL4.1/8/9,Pa PP2C11,Pa Sn RK2,and Pa ABF2 were significantly increased during apricot fruit ripening(p < 0.05).The expression levels of Pa PYL1/4.2 and Pa PP2C33 were also increased during the process,but there was no significant difference(p ≥ 0.05).On the contrary,the expression levels of Pa PYL2 and Pa PP2C27 were down-regulated,and no significant difference was observed.4.The full length of Pa PYL1,Pa PYL2,Pa PYL4.1,Pa PYL4.2,Pa PYL8,and Pa PYL9 were cloned using rapid-amplification of c DNA ends(RACE)method,and complete open reading frames of these gene contained 636 bp,600 bp,660 bp,648 bp,576 bp and 552 bp,and encoded 183,191,219,215,211,and 199 amino acids,respectively.Phylogenetic analysis showed that Pa PYL8 and Pa PYL9 clustered in the first subfamily,Pa PYL4.1 and Pa PYL4.2 belonged to the second subfamily,and Pa PYL1 and Pa PYL2 belonged to the third subfamily.Analysis of the protein domain showed that all six PYLs genes contained PYL＿PYR＿RCAR＿like highly conserved domains that interacted with other proteins,and there were four conserved loops CL1-CL4.The tertiary structure prediction found that Pa PYL1/4.1/8/9 were monomeric,while Pa PYL2/4.2 were dimer.5.Pa PYL9 gene was overexpressed in Tom tomatoes.Compared with wild-type tomato fruits,overexpression of Pa PYL9 promoted the coloration and softening of tomato fruits.CIRG of tomatoes increased significantly,hardness decreased rapidly.The contents of TSS increased significantly but TA decreased sharply(p < 0.01),indicating that the Pa PYL9 gene could promote fruit ripening.6.Transcriptome analysis of transgenic tomato showed that overexpression of Pa PYL9 significantly up-regulated the expression levels of ripening regulatory factors,such as ABA synthesis,ethylene synthesis,NAC,pigment synthesis and metabolism,fruit softening and flavor formation related genes,which suggested that Pa PYL9 could accelerate fruit ripening through its signal transduction pathway and integration of ethylene signal and other ripening regulatory factors.