Molecular Mechanism Of MdWUS2 Mediating Cytokinin Inhibition Of MdTCP12 In Regulating Axillary Bud Outgrowth In Malus

Nursery trees are the bases of the development of apple industry.Shoot branching is an important feature of plant architecture and flowering.Planting well-branched,rather than single-stem,nursery apple trees greatly contributes to early yield and production.However,the cv.’Fuji’,which is planted about 75%of cultivated area in China,natural branching is poor and this has been one of the problems demanding prompt solution in restriction of the development of apple industry in our country.Therefore,the research on the regulation mechanism of shoot branching is important for apple molecular breeding and the improvement of apple branch quality.Lots of researchers have shown that,cytokinin（CK）can significantly promote axillary buds outgrowth,however,the molecular mechanism is still unclear.In this study,the 1-year-old‘Nagafu No.2’（a‘Fuji’cultivar）/T337/M.robusta Rehd.,a grafting combination widely produced in Chinese nurseries was as the main material.Combining the analyses of hormone effects,phenotype and physiological traits,and RNA-seq,we selected the branch-related genes MdTCP12 and MdWUSs as the pivotal candidates and analyzed their functions in restraining or promoting axillary bud outgrowth and the transcriptional regulation in response to CK,in order to provide a point of CK in bud regulation.The main results are documented as followings:1.The apple axillary buds may also be a primary source for the synthesis of CK,and the CK biosynthesis is required for the activation of bud break.The outgrowth of axillary buds in nursery apple trees were found to be stimulated by the exogenous application of6-BA,the outgrowth of axillary buds on decapitated shoots,however,can be significantly inhibited by the application of the inhibitor of CK biosynthesis（Lovastatin）.The meristems had vigorous split ability in BA-treated and decapitated buds.On the contrary,the anatomical structures of axillary buds after Lovastatin application were similar with the untreated-buds.CK component,ZR level was higher after 6-BA or decapitation treatments relative to that of untreated buds,and the expression levels of CK biosynthesis genes MdIPT and the CK degradation genes MdCKX were significantly up-regulated.However,the ZR levels in Lovastatin treated buds were obviously lower than that in decapitated buds and the transcript levels of MdIPTs and MdCKXs were down-regulated.These indicated that the axillary buds could not be activated since the bud CK biosynthesis was suppressed.2.Constructed the RNA-seq of axillary bud in 6-BA treated and untreated buds at 0 h,4h,and 96 h,differential expression genes（5450）involved in hormones,axillary meristem activity,cell cycle and cell growth in response to CK were selected and identified in the treatments of 6-BA and control.Results indicated that differential expression genes in cytokinin signal transduction,auxin export and axillary meristem activity were significantly up-regulated at 4 h,which occurred sooner than the activation of the cell cycle and cell growth related genes.Additionally,branch related gene which belong to the II type TCP gene family was significantly down-regulated in response to CK,and the deeply study on this type genes would abundant the molecular mechanism of CK regulation of lateral branch development.3.The function of apple MdTCP12 gene on controlling branching was to suppress.MdTCP12 and MdTCP18 were highly expressed in axillary buds;but they were significantly inhibited by CK and decapitation,respectively,however,up-regulated by the Lovastatin treatment.MdTCP12 and MdTCP18 all had the TCP domain and mainly located in nucleus.The quantification of GUS activity showed that MdTCP12 and MdTCP18 promoters were repressed by 6-BA treatment,but enhanced after Lovastatin and GA₃ treatments,respectively.The overexpression of MdTCP12 transgenic Arabidopsis lines showed decreased number of rosette branches phenotypes compared to the wild type.The overexpressed MdTCP12 in Arabidopsis max2 mutant could reduce the abundant number of branches of max2 mutants and recovered to the WT type,partly.4.Apple MdWUS1 and MdWUS2 genes could trigger axillary bud outgrowth significantly.MdWUS1 and MdWUS2 were lowly expressed in inactive axillary buds;but they were significantly activated by CK and decapitation,respectively,and down-regulated by the Lovastatin treatment.MdWUS1 and MdWUS2 all had the conserved Wus-type domain and EAR motif（Lx Lx Lx）,and located in nucleus.The quantification of GUS activity showed that MdWUS2 promoter activity was induced by CK,but suppressed by Lovastatin.The overexpression of MdWUS2 transgenic Arabidopsis lines showed increased number of rosette leaves and branches phenotypes compared to WT,and the transgenic MdWUS2 tomato and tobacco lines were also exhibited increased branching phenotypes.Additionally,the overexpression of MdWUS2 transgenic Arabidopsis lines showed increased number of rosette leaves and branches phenotypes compared to WT.These suggested that MdWUS2（MdWUS1）which were activated by CK is a positive regulator of apple shoot branching.5.MdWUS2 could interact with MdTCP12 and repress the transcriptional activation activity of MdTCP12 targeted dormancy gene MdHB53b.The yeast two-hybird（Y2H）assay revealed that the EAR-motif containing C-terminal of MdWUS2 interacted with co-repressor MdTPL9,raising the possibility that MdWUS2 may function as transcriptional repressor.Additionally,the Y2H,Bi FC,and Co-IP assays confirmed the interaction between MdWUS2and MdTCP12.The RNA-seq showed that two apple dormancy genes MdHB53a and MdHB53b were obviously down-regulated in 6-BA treated buds.The GUS activity of MdHB53b promoter assay performed in Nicotiana benthamiana leaves showed that the promoter of MdHB53b was transcriptional activated by MdTCP12,however,the transcriptional activity of MdTCP12 was largely suppressed by MdWUS2 in regard to the activation of MdHB53b transcription.Taken all these results together,we proposed a model for MdWUS2 mediation in CK inhibition of MdTCP12 regulation of axillary buds outgrowth.In the inactive buds,the expression of MdWUS2 was inhibited.However,the MdTCP12 were expressed highly in axillary buds particularly,and promoted the expression of bud dormancy gene MdHB53b and inhibited the outgrowth of axillary buds.When the CK was synthesized or increased,the MdWUS2 were activated obviously.The EAR-motif containing MdWUS2 could recruite the transcriptional co-repressor MdTPL9 protein,and interact with MdTCP12 to suppress its activity.Therefore,the bud dormancy gene MdHB53b was down-regulated and shoot branching was promoted.