The Effects Of Exogenous Glucose On Apple Flower Bud Differentiation And Function Analysis Of Candidate Gene MdSC35₂

Sugar,as an important energy and signal substance,plays an important role in the flowering process of woody fruit trees.Exogenous glucose spraying treatments have an obvious promotion to the flowering of Fuji apple varieties,but the molecular mechanism by which glucose regulates flower bud differentiation is unclear.Therefore,it is of great theoretical and practical significance to explore the key regulatory pathways and candidate genes for floral formation induced by glucose treatment in Fuji apple,so as to guide high quality and efficient production of apples and to understand the regulatory theory of apple flower differentiation.In this study,the weakly Fuji apple flowering cultivar�Nagafu NO.2'was used as the research material.Firstly,an exogenous glucose treatment was conducted and its promotion of flowering-related phenotypes was clarified.Succeeding,the key regulatory pathways and networks in response to glucose treatment induced floral formation were comprehensively identified in combination with RNA-seq data of spur terminal buds during floral induction stage.The key candidate gene MdSC35?2 was obtained,and the biological function of its regulation on flower formation was clarified,meanwhile,its interaction proteins were screened.The regulational molecular mechanisms of MdSC35?2on apple flowering via glucose and ABA signaling were preliminarily analyzed.The main results are as follows:1 The molecular mechanism underlying glucose-promoted floral differential was constructed.A low concentration glucose solution?1.5%?treatment to�Nagafu NO.2'apple trees at 28,30 DAFB?days after full blooming?obviously accelerated the bud growth and floral induction and significantly increased the flower formation rate in the coming year.The content of endogenous sugar and hormone in buds was remarkably affected,showing increasing changes of glucose,sucrose,sorbitol and IAA,and a decreasing change of GA₄.RNA-seq data displayed a total of 12059 non-redundant DEGs?differential expression genes?during floral induction stage.These DEGs were noticeably enriched in the pathways related to carbon metabolism,hormone synthesis and signaling,and photosynthesis,and presented expression profiles that are conducive to flower induction.More than 770 TFs?transcription factors?responded to glucose treatment,among them,some TF families related to cell fate determination,embryogenesis,meristem maintenance,and flower development regulation were significantly up-regulated,and multiple flowering genes also were promoted in gene expression.15 splicing factors responsed significantly to the glucose treatment,among them,4 MdSR proteins were completely down-regulated in expression.And these splicing components may regulate apple bud differentiation via glucose signaling.2 The MdSR protein family in apple was systematically analysed.36 MdSRs were indentified and were divided into seven subfamilies including MdSC,MdSCL,MdSR,MdRS,MdRSZ,MdRS2Z and MdSR45 according to the structural characteristics.These proteins had similar physicochemical properties and generally discontinuous gene structures.Although the molecular weight of MdSRs varied from 10.27 k Da to 59.56 k Da,all members had the iconic RRM domain and RS domain.The ubiquitous cis-acting elements in response to hormones and environmental signals,such as Me JA,ABA,light and low temperature,were common in the upstream promoter of MdSRs,suggesting that this family is widely involved in environmental response?especially stress response?in apple.Most MdSRs were highlier expressed in roots,stems and buds,indicating that they were mainly functioned in the development of those apple tissues.Glucose treatment significantly down-regulated MdSR gene expression according to the RT-q PCR data,but all MdSRs expressions increased along with floral induction,comprehensively reflecting the crucial role of MdSRs in apple floral differentiation,and MdSRs could be involved in glucose-mediated floral induction.MdSC35?2 was selected as candidate SR genes for further functional analysis.3 The candidate gene,MdSC35?2,was isolated from�Nagafu NO.2',and its gene structure was highly conserved and localized in the nucleus.There was negative feedback regulation between MdSC35?2 and glucose.Exogenous glucose treatment resulted in a decrease in MdSC35?2 gene expression in apple buds.Overexpression of MdSC35?2motivated the utilization of glucose by apple callus cells and thus the growth vigor of transgenetic callus was better than the wild type.It was also found that MdSC35?2 can obviously promote the expression of sugar signal genes MdHXK2 and MdKIN10independently of glucose concentration.MdSC35?2 over-expressing tomato lines repersented the early flowering phenotype and the significant up-regulation of flowering genes and Sl HXK.All of these suggested that MdSC35?2 may regulate cell growth and flower formation by upregulating glucose signal genes MdHXK2 and/or MdKIN10.In addition,this work also found that MdSC35?2 and ABA signals promoted each other and may work coordinately to regulate cellular stress tolerance.According to the results of yeast two-hybrid,MdSC35?2 mainly affected the function of specific genes through splicing regulation.In addition,interaction with MdCYP59 indicated that MdSC35?2 may also function in gene transcription regulation.