Mechanism Analysis Of Phytohormone Regulates Seed Abortion And Fruit Development Of Seedless Pear

As one of the origins of pears,China has a wide range of planting areas and abundant germplasm resources.At present,there have been many reports on the mechanism of seedless fruits,but there are few reports on the molecular mechanism of seed death and seedless fruit development in seedless pear.In this study,‘1913'seedless pear(Pyrus spp.)was used as material to identify the types of seed abortion of‘1913'pear and the functions of various hormones activated by pollination on seed death and seedless fruit development.Analyzing the mechanism of ethylene and jasmonic acid(JAs)regulating the death of‘1913'pear seeds,and the molecular mechanism of PbGA20ox2 and PbGIF1 gene respectively mediating gibberellin(GA)and cytokinin to regulate the development of pear fruit,and preliminarily analyzed the mechanism of hormones in regulating seed abortion and fruit development of seedless pears.It provides a theoretical basis for the research on the development of seedless fruits and the selection and breeding of seedless varieties.The main findings are as follows:1.The fertilization disorder caused the abortion of‘1913'pear seeds.Using‘1913'pear as the material,and through different pollination treatments,observation of floral organ morphology,identification of chromosome ploidy,identification of the male and female gametes fertility,observation of the early development of ovules and the fertilization process,we determined that the type of seed abortion in‘1913'pear is stimulating-type parthenocarpy,and the development of its fruit depends on pollination.In addition,the ovules of‘1913'pear begin to abort on the 7th day after pollination,and the seeds are completely dead on the 12th day.Further analysis confirms the failed fusion of sperm and egg cells is the basic reason of seed abortion in‘1913'pear.2.Ethylene and JAs activate the expression of senescence-related genes to regulate the death of unfertilized ovules in‘1913'pear.Through the determination of endogenous hormone content in the pollinated ovules and the analysis of transcriptome data,we found that pollination activates the synthesis of multiple hormones in the ovule.Among them,the fertilization disorder leads to the excessive accumulation of ethylene in the ovule of‘1913'pear.The results of exogenous ACC treatment of isolated pear seeds showed that ethylene positively regulated the death of pear seeds.The functional verification assay of the ETHYLENE INSENSITIVE 3-LIKE(Pb EIL1)gene,which encodes a transcription factor that mediates ethylene signaling,showed that the specific overexpression of the Pb EIL1 gene in tomato seeds leads to premature senescence of the seeds.Screening the downstream target genes of Pb EIL1 and throughing a series of interacting assays,we found that Pb EIL1 can directly activate the transcription of the ovule-specific expression gene SENESCENCE-ASSOCIATED CYSTEINE PROTEINASE 1(Pb Cysp1)and to promoting the senescence of‘1913'pear ovules.In addition,the application of GA₄₊₇on the unpollinated ovary caused the death of‘1913'pear ovules,on the contrary,it inhibited the senescence process in unpollinated ovules of‘Bartlett'and‘Dangshansu'.Analysis of endogenous hormone content showed that ethylene,JAs and salicylic acid(SA)in‘1913'pear ovules were higher than that in other two varieties.An in vitro experiment proved that the exogenous ACC,JAs and SA treatments promoted the death of pear seeds.And a further research found that GA₄₊₇ treatment promoted the synthesis of endogenous ethylene and JAs in the ovules of‘1913'pear,but did not affect the content of SA in the ovules.q RT-PCR experiment showed that the expression of ethylene and JAs pathway-related genes in different regions of the unpollinated ovule showed higher expression in the dead part,while the expression of SA-related genes did not differ significantly in different regions of the ovule.Further studies on the mechanism of JAs regulating ovule senescence showed that JAs signal responder Pb MYC2 can directly activate the transcription of death-related gene SENESCENCE-ASSOCIATED GENE 39(SAG39)to inducing the death of‘1913'ovules.3.PbGA20ox2 promotes the synthesis of GA₄ and mediates GAs to regulate fruit development of seedless pear.The development of‘1913'pear fruit depends on pollination.Pollination activates the synthesis of GAs in‘1913'pear fruit and the application of exogenous GA₄₊₇ induced the formation of seedless fruit in‘1913'pear.Basing on the transcriptome data,we screened out the key gene GIBBERELLIN 20-OXIDASES(GA20oxs),which regulates the synthesis of GAs,and identified three GA20oxs genes in pear genome:PbGA20ox1-3.By analyzing the expression patterns of three PbGA20oxs genes,we confirmed that PbGA20ox2significantly responds to pollination signals and is related to the early development of pear fruit.The functional verification assay of PbGA20ox2 suggested that PbGA20ox2 promotes the development of transgenic tomato seedless fruit by up-regulating the synthesis of GA₄,but not GA₃ and GA₁.In addition,the transient overexpression of the PbGA20ox2 in the pear ovary promoted the synthesis of GA₄ in the pear ovary and regulated the fruit setting and the unpollinated ovary development.Taking together,PbGA20ox2 promotes the development of seedless pear fruits by promoting the synthesis of effective gibberellin GA₄,which is required for fruit development of seedless pears,instead of GA₃ and GA₁.4.PbGIF1 promotes cell proliferation and mediates the regulation of cytokinins on the development of seedless pear fruit.Pollination activates the synthesis of cytokinins in the‘1913'pear fruit,and cytokinins homolog CPPU treatment induced the seedless fruit development in‘1913'and‘Dangshansu'pear.During the early stage of pear pollinated-ovary development,the content of cytokinins increased first and then decreased.The results of exogenous CPPU treatment assay showed that CPPU promotes pear fruit development by promoting cell division in the epicarp and mesocarp.According to the transcriptome data,we screened out a transcriptional coactivator GRF-INTERACTING FACTOR1(GIF1)which is closely related to cell division.By analyzing the expression pattern and the function of PbGIF1,we found that PbGIF1 is highly expressed in pear hypanthium and the overexpression of PbGIF1 promotes the tomato fruit development and the growth of pear flesh calli by promoting cell proliferation.Further studies have found that the cytokinins signal responder Pb RR1 can directly activate the transcription of PbGIF1 and promote pear fruit development.In addition,PbGIF1 can directly bind to Pb RR9 protein to enhance the transcriptional activation of Pb RR9 to its downstream gene Pb YUCCA4,thereby promoting the development of pear fruit.In summary,fertilization disorder is the basic cause of the abortion of‘1913'pear seeds.Pollination activates the synthesis of multiple hormones in the ovules and fruit of‘1913'pear,and the hormones participate in regulating the seed abortion and fruit development in‘1913'pear.Among them,the ethylene signal mediator Pb EIL1 directly activates the transcription of Pb Cysp1 to mediate the regulation of ethylene on the‘1913'pear ovule abortion,and the JAs signal responder Pb MYC2 activates the transcription of Pb SAG39 to mediate the death regulation of the‘1913'pear ovule abortion by JAs.In addition,PbGA20ox2 promotes the development of pear fruit by regulating the synthesis of GA₄.Moreover,PbGIF1 is directly regulated by Pb RR1 or directly combined with Pb RR9 to participate in the process of cytokinin regulating the development of pear fruit.