The Mechanism Of GA₃ And GA₄ Differentially Induced Parthenocarpy In Pear

Most pears are naturally self-incompatible,which need artificial pollination to improve fruit set for ensuring its yield.But artificial pollination is wasting time and labour,thus increasing the cost of pear production.Parthenocarpy refers to the phenomenon that ovaries can directly develop into matured fruits by uncoupling pollination and fertilization,which is a good approach to release the restrict that the fruit set bring to pear fruit production.In this study,‘Dangshansu'pear belonging to White pear variety and‘Starkrimson'pear belonging to Eurpean pear are used as experimental materials to explore the mechanism of the difference between GA₃-and GA₄-induced parthenocarpy by spraying GA₃ and GA₄ at full bloom stage.The main research results are as follows:1. GA₄ prior to GA₃ induces parthecocarpy in‘Dangshansu'pear.GA₄ is better to induce pathenocarpy than GA₃ in‘Dangshansu'pear.When the fruit-lets of unpollinnation treatment fully drop off,the fruit set rate of GA₄ treatment reached 96.8%,which was significant higher than 56.8%of GA₃ treatment.At the same time,the fruit set rate of pollination treatment was 84.3%.Fruit-lets treated with GA₄ can harvested seedless fruits at 135 DAA?days after anthesis?,which treated with GA₃ fully fall off at 40 DAA thus could not grow into matured fruits.At 6 DAA,Young fruits treated with GA₄ were significantly larger than those treated with artificial pollination and GA₃.Histological observation of paraffin sections showed that no matter the inner calyx or the external calyx of fruit-lets treated by pollination were thicker than that of those treated by un-pollination,where the cell layers differed in significance.Compared with GA₃ treated fruit-lets,GA₄-treated young fruits significantly increased both the thickness of inner calyx and the outer calyx by promoting the cell division of the outer calyx and the cell expansion of the inner calyx,thus increased the total thickness of the calyx.The content of ABA levels were common decreased in fruit-lets treated by GA₃ and GA₄.The endogenous IAA content was increased in GA₄ treated fruit-lets,which was significant than GA₃ treated fruit-lets.Thus we speculated that GA₄ prior to GA₃ induced better parthenocarpic fruit set by increasing the IAA content in‘Dangshansu'pear.2. Differentially effects of GA₄ and GA₃ on the genes related to cell development and hormones in‘Dangshansu'pear.Genes directly related to the cell development and hormone pathway were identified and screened through transcriptome.Results showed 48DEGs?Differential Expressed Genes?related to cell division,14 DEGs directly related to cell expansion were screened.Exogenous GA₄ inducedmore DEGs related to cell development.By q-PCR,key genes related to fruit development were identified including Pbcyclin-dependent kinase B2-2?Pb CDKB22?Pbcyclin-dependent kinase B2-2-like?Pb CDKB22L?,Pbexpansin-A4?Pb EXPA4?,Pb Cyclin A2-4?Pb CYCA2-4?,Pb Cyclin B2-4?Pb CYCB2-4?and Pbcyclin-dependent kinase inhibitor 6-like?Pb CDKI6L?.By applying transcription analysis to four treatments of fruitlets at 4 DAA and screening the DEGs related to hormone pathway,we found no matter GA₃ and GA₄ can provoked the gibberellins signaling and repressed the ABA metabolic pathway,while GA₄ was superior to GA₃ in activating the differential expression of auxin synthesis rate-limiting enzyme gene Pb YUCCA6.Taken together,GA₄ prior to GA₃ activated the synthesis of auxin and the expression of relative genes,thus leading to better fruit set and development in‘Dangshansu'pear.3. GA₃ prior to GA₄ induces parthecocarpy in‘Starkrimson'pear.Spraying GA₃to the flowers at full bloom stage could induce the fruit set 42.9%at almost 20 DAA,which was significantly higher than 15.9%,the fruit set rate of GA₄ treatment.The fruit set rate of hand pollination reached 40.3%.After 100 DAA,un-pollinated fruit-lets treated by GA₃could develop into parthenocarpy,but un-pollinated fruit-lets treated by GA₄ completely shed at 35 DAA.At 6 DAA,fruit-lets of GA₃ treatment and GA₄ treatment were both enlarged and were significantly bigger than those of un-pollination treatment.Histological observation of paraffin sections showed that GA₃ treated fruit-lets and GA₄ treated fruitlets had significant cell division and cell expansion compared with un-pollinated treatment.However,the thickness of the calyx after GA₃ treatment was significantly thicker than that of GA4 treatment,which was attributed to the increase of the thickness of the inner calyx and the outer calyx.Cell area analysis showed that GA₃ was better than GA₄ in promoting cell division of inner calyx and cell expansion of outer calyx.Through the quantification of hormone,we also found GA₄ was better than GA₃ in promoting the synthesis of IAA level,but GA₄ treatment could not reduce the level of ABA in‘Starkrimson'fruit-lets.Therefore,we speculate that GA₃ was superior to GA₄ in reducing ABA content,thus inducing better cell division and cell expansion leading to better fruit setting.4. Differentially effects of GA₄ and GA₃ on the genes related to cell development and hormones in‘Starkrimson'pear.We applied transcription analysis to four treatments of fruitlets at 4 DAA and screened genes directly related to the cell development and hormone pathway through transcriptome.Results showed 28 DEGs related to cell division,7 DEGs directly related to cell expansion were screened.Exogenous GA₄ induced more DEGs and higher expression of genes related to cell development.By q-PCR,key genes related to fruit development were screened.Pc EXPA4?Pc Cyclin A2-4?Pc CDKB22 were identified to accord with the trend of fruit development and exogenous GA₃ prior to GA₄ induced the expression of Pc EXPA4?Pc Cyclin A2-4?Pc CDKB22.Through screening the DEGs related to hormone pathway,we found GA₄ can stimulated the differentially expression of the members of auxin synthesis rate limiting enzyme gene,but GA₃ did not have the ability.Through the analysis of ABA metabolism,GA₃ treatment could significantly inhibit the expression of ABA synthesis key gene Pc NCED1?9-cis-epoxycarotenoid dioxygenase?.The result could not induced by GA₄treatment.The results above showed that GA₃ was superior to GA₄ in inhibiting the expression of Pc NCED1 and the expression of cell reproduction related genes thus induced higher parthenocarpic fruit set and fruit development in‘Starkrimson'pear.5.Identification and functional analysis of fruit set related genes downstream of hormoneThrough the comparing DEGs from four different treatment group Pollinated vs.Unpollinated;Parthenocarpic treatment vs.Non-parthencarpic treatment),Pb CYP78A6L was identified.We cloned Pb CYP78A6L and predicted its function.Pb CYP78A6L is closely related to At CYP78A6 and At CYP78A9 in arabidopsis.The expression pattern of Pb CYP78A6L in early stage of pear fruit-lets was similar to that of key genes during fruit development.The expression of Pb CYP78A6L was highest in sepals by Tissue specific expression,its promoter could directly respond to the induction of exogenous active gibberellin,and the protein encoded by Pb CYP78A6L gene was localized on the membrane.Pb CYP78A6Lacted downstream of hormone and upstream of cell development to regulate fruit set and fruit development of pear.