Molecular Mechanism Of Sucrose Regulates Axillary Bud Growth In Chrysanthemum

Chrysanthemum(Chrysanthemummorifolium),as one of the ten famous flowers originated in China and the four cut flowers in the world,possesses high ornamental and great economic value.However,in the process of single-headed cut chrysanthemum production,it takes a lot of manpower and material resources to manually wipe out the axillary buds.This labor-intensive production method limits the development of the chrysanthemum industry to a certain extent.Sucrose can promote the axillary bud outgrowth in plant,but the effect of sucrose in shoot branching of chrysanthemum has not been reported.The aim of this study was to investigate the effect of sucrose in axillary bud growth of chrysanthemum and to find key genes regulating this process.Using molecular biology methods to study the molecular mechanism of axillary bud outgrowth of chrysanthemum from the genetic level,to improve the plant shoot branching regulation network and laid the foundation for cultivation of new varieties of chrysanthemum that meet the needs of various plant types.The main contents and conclusions are as follows:1.Here,after the treatment of two-node stems isolated from chrysanthemum with 0 mM and 20 mM sucrose in the dark,20 mM sucrose was found to promote the growth of only the top bud.The detection of 14C-sucrose radioactivity demonstrated that sucrose was transported to the growing top bud.Based on phenotypic differences,RNAseq results,gene expression quantification and auxin content analysis,sucrose treatment upregulated the auxin transport carrier CmPINl and CmAUX1;IAA content decreased and promoted auxin export from buds;upregulated the inhibitory auxin signaling genes CmIAA3,CmIAA14,and CmIAA16;downregulated the auxin response factor CmARF2 in the growing top bud.The administration of CCCP(sucrose transport inhibitor)or TIB A(auxin transport inhibitor)or IAA together with 20 mM sucrose weakened the promoting effect of sucrose in top bud outgrowth.Auxin-related gene expression analysis and auxin content measurements revealed that in the top bud of the CCCP or TIBA or IAA treatment,CmPINl and CmAUX1 were downregulated;auxin export from the bud was inhibited and IAA content increased;the expression of CmIAA3,CmIAA14,CmIAA16 and CmARF2 was impacted.In conclusion,sucrose was transported into buds to promote bud outgrowth,possibly through an auxin transport pathway-dependent mechanism.2.In the transcriptome analysis,it was found that the dormancy-associated gene CmDRM1 was down-regulated in the growing top bud after sucrose treatment,which was consistent with the expression trend of CmARF2.CmDRM1 was cloned to a full length of 336 bp,encoding 111 amino acids.The phylogenetic tree showed that CmDRM1 is closely related to Arabidopsis AtDRM1.Subcellular localization and transcriptional activity experiments indicated that CmDRM1 protein is localized in the nucleus and has transcriptional activation activity.Tissue quantification showed that CmDRM1 was expressed higher in leaves and stems,followed by roots and lateral buds,and lower expression in apical buds.The CmDRM1 promoter was cloned to obtain 2367 bp,and the elemental analysis revealed that the promoter of CmDRM1 contained multiple ARF binding elements(AuxRE),and CmDRM1 was induced by auxin in chrysanthemum leaves and lateral buds.Further yeast one-hybrid experiments showed that the auxin response factor CmARF2 binds to the CmDRM1 promoter.Therefore,combined with the results of transcriptome analysis,it is demonstrated that CmDRM1 is involved in the promotion of sucrose in axillary bud growth of chrysanthemum,and there may be a regulation pathway of axillary bud growth of "sucrose-auxin-CmDRM1".3.CmDRM1 was separately transformed into the chrysanthemum 'Jinba' and Arabidopsis.It was found that the transgenic chrysanthemum interfering lines of CmDRM1 showed a phenotype that promoted axillary bud growth and increased shoot branching,which further confirmed the possible existence of a "sucrose-auxin-CmDRM1" axillary bud growth regulation pathway.Our research group found that CmTPL1.1 has a multi-branched phenotype after heterologous transformation into Arabidopsis,which is identical to the transgenic Arabidopsis phenotype of CmDRM1.Using yeast two-hybrid and tobacco BiFC experiments,verification showed that CmDRM1 can interact with CmTPL1.1.Therefore,the results indicate that CmDRM1 may regulate plant shoot branching through CmTPL1.1.4.Based on the phenotypic difference after the sucrose treatment of two-node stems,quantitative analysis of sugar-related genes revealed that CmSWEET17 was highly expressed in the growing top axillary bud,indicating that CmSWEET17 may be involved in the promotion of sucrose in axillary bud growth of chrysanthemum,which is positively correlated with axillary bud growth.The fructose transport protein CmSWEET17 was cloned to a full length of 876 bp,encoding 291 amino acids;sequence analysis revealed that CmSWEET17 contains six transmembrane domains belonging to the Clade ?subfamily in the SWEET family;subcellular localization analysis revealed that CmSWEET17 is localized on the cell membrane.The quantitative analysis showed that CmSWEET17 had the highest expression in leaves,followed by stems,and the roots were extremely low.In addition,CmSWEET17 was induced by sucrose in chrysanthemum leaves and axillary buds.The overexpression lines of CmSWEET17 were obtained by transgenic technology,and after the two-node stems experiment,it was found that the overexpressing lines of CmSWEET17 promoted the axillary bud growth.Quantitative analysis revealed that the expression levels of auxin transport proteins CmAUX1,CmLAX2,CmPIN1,CmPIN2 and CmPIN4 were increased in the overexpressing line,and IAA content of axillary bud decreased.Therefore,it was suggested that CmSWEET17 may promote the axillary bud growth by regulating the transport of auxin.