Molecular Mechanism Of Abnormal Physiological Fruit Drop In Guizhou Sweet Cherry

Sweet cherry(Prunus avium L)belongs to Prunus of Rosaceae,which is native to western Asia and southeastern Europe.It is widely planted in China,the United States,Turkey,Chile and other countries.It is known as a treasure in fruit because of its early maturity,rich nutrition,bright fruit color and excellent taste.In recent years,it has been widely introduced in southern China and planted in a large area in central and western Guizhou,but serious physiological fruit drop is common Bottlenecks.Revealing the mechanism of abnormal physiological fruit drop of Southern sweet cherry has important guiding significance for improving fruit setting rate.In this study,sweet cherries planted at medium and high altitude in Guizhou were used as materials to analyze the mechanism of abnormal physiological fruit drop of sweet cherries in Guizhou from the physiological,cellular and molecular levels,in order to provide theoretical reference for improving the yield of sweet cherries in South China.The main results are as follows:1.The abnormal physiological fruit drop characters of sweet cherry in GuizhouThe pollen viability,pollen morphology,embryo viability,embryo morphology,fruit drop rate,the anatomical structure of abscission zone,carbohydrate content,enzyme activity and phytohormone content of carpopodium of sweet cherry varieties in Guizhou were observed and measured.The results showed that the pollen viability of“Santina”,“Brooks”,“Sparkerry”and“Black Pearl”was lower,and the rate of pollen deformity was higher,while that of“Qianying No.1”was higher,and the pollen morphology was regular and oval,and the rate of pollen deformity was low.The fuller the embryo,the higher vitality of the embryo,and the less likely it is to drop fruit.The fruit drop rates of“Santina”,“Brooks”,“Sparkerry”and“Black Pearl”were all more than 89%,and the fruit drop rate of“Brooks”was the highest(97%).In the two peak periods of fruit drop,the abscission zone of the normal carpopodium began to form,but it was not obvious enough,while the abscission zone of the abscising carpopodium had been completely formed.The glucose content of the fruit and its carpopodium were lower than that of the normal fruit and its carpopodium,but there was no significant difference between fructose and sucrose.The activities of cellulase(CEL),Pectinase(PE),polygalacturonase(PG)and peroxidase(POD)in the abscising carpopodium were significantly higher than those in the normal carpopodium,and those in the abscising fruit also significantly higher than those in the normal fruit.The contents of auxin(IAA,IBA),gibberellin(GA₃,GA₇),cytokinin(TZ),1-aminocyclopropanyl-1-carboxylic acid(ACC),jasmonic acid(JA)and methyl jasmonate(Me JA)in the abscising pedicel were significantly lower than those in the normal carpopodium,while the content of abscisic acid(ABA)was significantly increased.2.Transcriptome analysis of the abscising carpopodium in sweet cherryCompared with normal carpopodium,auxin biosynthesis genes(?-Trp,ami E,YUCCA)and auxin signal transduction genes(AUX1,AUX/IAA,ARF)were significantly down-regulated,while ethylene biosynthesis related genes(ACS,ACO),ethylene signal transduction related gene(ERF)and abscisic acid biosynthesis gene(NCED)were significantly up-regulated.The expression of cellulase(CEL),pectinesterase(PE),polygalacturonase(PG)and?-galactosidase(?-gal)genes were significantly up-regulated,and the expression of the peroxidase(POD)gene was also significantly up-regulated.The Expansin(EXP)and xyloglucan endotransglucosylase/hydrolases(XTH)genes were also significantly up-regulated.However,the genes involved in cytoskeleton formation(MAP,?-TUB and ACT),were significantly down-regulated in the abscising carpopodium,which indicated that in the abscising carpopodium,the cytoskeleton formation was inhibited,the new cells could not be formed normally,and the programmed cell death(PCD)occurred in the old cells,which led to the shedding of plant organs.In addition,compared with the normal carpopodium,the transcription factors(MYB,b HLH,WRKY,HD-ZIP,MADS,ERF,NAC and b ZIP)may be involved in fruitlet shedding of sweet cherry were also significantly up-regulated in the abscising carpopodium.3.Proteome analysis of the abscising carpopodium in sweet cherryCompared with normal carpopodium,the auxin biosynthesis and signal transduction related proteins(?-Trp,AUX1,TIR1)were significantly down-regulated,while ethylene synthesis-related proteins(ACO),abscisic acid synthesis-related proteins(NCED),and abscisic acid signal transduction related proteins(PP2C,Sn RK2)were significantly up-regulated.Additionally,the expression of cell wall modification-related proteins(CEL,PE,PG,?-gal,EXP,XTH)was also significantly up-regulated in the shedding carpopodium,while the cytoskeleton biosynthesis-related proteins Arp2/3 and microtubule-related protein(MAP)were significantly down-regulated,and the Pav HB16,a transcription factor of the HD-ZIP gene family was also significantly up-regulated in the abscising carpopodium.4.Identification and expression analysis of HD-ZIP gene family in sweet cherryAccording to the genome-wide identification of the HD-ZIP gene family in the sweet cherry,it was found that there were 27 members in the sweet cherry HD-ZIP gene family,which were divided into four subfamilies.The 27 genes are unevenly distributed on 8 chromosomes of sweet cherry.Compared with other tissues,the Pav HB2 was higher in normal fruits during the first and second peak fruit drop periods.Besides,Pav HB12 was significantly up-regulated in normal carpopodium during the two peak fruit drop periods.Compared with normal tissues,both Pav HB16and Pav HB18 were up-regulated in abscising tissues.The result showed that the complete open reading frame(ORF)of the Pav HB16 contained 668 bp,encoding 219 aa,and the protein encoded by this gene was expressed in the nucleus.Through the cloning and element analysis of the Pav CEL18 promoter,the results showed that there were two elements(AAATTAAA)that can be bound by the HD-ZIP I transcription factor.The result indicates that the Pav CEL18 may be regulated by Pav HB16 protein.The results of Arabidopsis thaliana transformed with Pav HB16 showed that the Pav HB16 could promote the expression of abscission-related genes(At CEL3,At PG1 and At EXPA24),moreover,the Pav HB16 also promote the development of abscission zone and the shedding of floral organs in Arabidopsis thaliana.5.Cloning and expression analysis of functional genes related to shedding in sweet cherryThe functional genes related to sweet cherry shedding were cloned,moreover,the bioinformatics and expression analysis were performed on them.Among these genes,the cellulase gene(Pav CEL18)was 1,704 bp,and the ORF was 1,494 bp,encoding 497 aa,with glycosyl hydrolase active sites.The phylogenetic tree showed that Pav CEL18 was closely relative to the CEL proteins from Prunus mume,Prunus persica and Prunus dulcis.The expression level of Pav CEL18in the abscising organs was significantly up-regulated.In addition,we also obtained polygalacturonase(Pav PG)gene,the gene was 1,644 bp,and the ORF was 1,443 bp,encoding 480aa.The phylogenetic tree showed that the Pav PG of the sweet cherry has a higher homology similar to that in Prunus persica and Prunus dulcis,and it is significantly up-regulated in the abscising tissues.Besides,the expansin(Pav EXPA2)gene was 1,035 bp,and the ORF was 852 bp,encoding283 aa.The phylogenetic tree showed that the Pav EXPA2 of the sweet cherry has a higher homology similar to that in Prunus mume,Prunus dulcis,and Prunus persica,and it significantly up-regulated in the abscising tissues.Additionally,the ACC oxidase gene(Pav ACO)was 1,175 bp,and the ORF was 960 bp,encoding 319 aa,and the expression level of Pav ACO was also significantly up-regulated in the abscising tissue.In summary,this study found that the mechanism of abnormal physiological fruit abscission of Guizhou sweet cherry was mainly due to the reduction of auxin synthesis in vitro caused by embryo abortion,the increased sensitivity of abscission zone cells to ethylene,and then promote the expression of shedding related genes.Under appropriate temperature and p H,the activity of cell wall degrading enzymes increases,degrade cellulose and pectin in the cell wall,and then cell separation and fruit drop were promoted.