Functional Study Of MAX2 Gene On The Regulation Of Axillary Bud Outgrowth By Mediating Strigolactone Signaling In Malus

The breeding of large seedlings with branches is the developmental direction of apple seedling in the world,but the seedlings are difficult to produce a certain number of branches.Seedling branch and plant type of apple tree are affected by the germination characteristics of axillary buds.In season,the axillary bud of apple usually could not germinate.Both the meristem formation,and sustained activity and growth are involved in the features of axillary bud outgrowth.Generally,plant branching is a quantitative trait regulated by multigenes,and easy affected by environmental conditions.Lots of studies have show that changes belonged to auxin,cytokinin（CTK）,strigolactone（SL）,gibberellin,and sucrose,can significantly affect branching number.There are some differences of these factors on the regulatory effect among species.Moreover,the molecular mechanisms are still unclear.More-branching mutant（MB）of Malus baccata and its wild-type（WT）in apple were mainly used as experimental plants,which were grafted on biennial baleng crabapples（Malus robusta）.Combining the analyses of phenotype,physiological and biochemical traits,hormone effects and RNA-seq,the physiological basis and molecular mechanism were identified for producing more branches.Using the expression pattern of MAX2 gene,cloning and identification of gene and promoter sequences,and genetic transformation of Arabidopsis,the function of MAX2 gene on regulating bud outgrowth was studied,which provide a theoretical basis for the regulated technologies of high quality branching seedlings in apple.The main results were obtained as follows:1.Obtained the physiological bases of phenotypic differences between WT and MB grafting seedlings,systematically.Axillary buds formed normally both in WT and MB,and no difference in bud number was recored.Plant phenotypes were similar in the initial stage of germination,and however,the outgrowth of axillary buds was firstly observed in MB at approximately 30 days after germination.The number of MB branches increased significantly to three times than that of WT.MB displayed the formation of secondary branches,relative to WT.Traits of less branch angle,shorter internodes,dwarfing,increased stem diameter were found in MB scion,which affected the root growth and development of rootstock.The hormone levels,photosynthesis efficiency,nutritient distribution and antioxidant capacity of MB grafted plants revealed that the growth viability and resistance were weak in MB.Based on above results and related literature studies,it was speculated that more branches of MB mainly came from the sustained outgrowth of axillary buds,and the reason of MB mutation was part of the key pathways responsible for regulating the outgrowth of axillary buds.2.The outgrowth of apple axillary buds was related to CTK,glycometabolism,auxin polar transport and SL.CTK component,ZR,was significantly higher,while IAA level was significantly lower in MB buds relative to WT.There were no differences between WT and MB on gibberellin contents,involving GA₁₊₃ and GA₄₊₇.Levels of soluble sugar containing sucrose,glucose,fructose,and sorbitol were significantly higher in MB than that of WT.Exogenous cytokinin stimulated the outgrowth of MB buds.Sucrose levels of WT buds,however,were apparently decreased in response to CTK application,relative to that of glucose and fructose,which were significantly increased.In addition,the outgrowth of WT and’Fuji Nagafu No.2’buds was not induced with sucrose application,which might state that sucrose was primarily served as nutrition in apple,rather than an activated signal for stimulating bud outgrowth.Bud outgrowth in both WT and MB was completely suppressed with the inhibitor,NPA,application of auxin polar transport on axillary buds,which suggested that bud outgrowth was relied on the auxin polar transport from axillary buds.Exogenous SL（GR24）could completely suppress the bud outgrowth of WT,T337,’Fuji Nagafu No.2’,M26,but not the MB buds.Moreover,the more branching phenotype of MB was maintained as grafted on normal stocks,which stated that the more branching phenotype of MB was particularly related to the SL signaling pathway.3.Constructed the RNA-Seq of formed axillary buds in WT and MB,and the analysis of differentially expressed genes showed that 728 genes were down-regulated and 388 genes were up-regulated in MB.These genes were significantly enriched in GO functional classifications related to the regulation of transcription and DNA-dependent,signal transduction,protein binding,ATP binding,and phosphorylative mechanism.KEGG pathways of these genes were significantly enriched in plant hormone signal transduction and RNA metabolism,and about 70%genes associated with hormone signaling and cell activity were down-regulated.Gene expression of hormone signal transduction pathway and outgrowth buds of different stages suggested that bud activation was positive to auxin polar transport,CTK signaling,and the vitality of sugar metabolism and transport.The patterns of increase and following decrease of strigolactone genes might be related to the feedback regulation mechanism involving in SL pathway.Referring to the phenotypic characteristics of SL-related mutants in model plants,it was suggested that MbMAX2 gene might be closely related to the production of MB phenotypes.4.The function of apple MAX2 gene on regualting branching was suppressing by mediating SL signaling.Based on the RNA-seq ananlysis and branching phenotypes,the expressions of MAX2（MDP0000305017）and D14（MDP0000529739）were significantly changed accompanying bud outgrowth.The expressions of apple MAX2 and D14 had no clear tissue specificity,and no correlation between bud outgrowth and expressional levels of MAX2and D14 was identified.Gene expressions of MbMAX2 and MbD14 were decreased after GR24 treatment.In addition,GUS activity of MbMAX2 and MbD14 genes was decreased after GR24 treatment.These results showed that SL could suppress the transcripts of MbMAX2 and MbD14.Transgenic Arabidopsis test showed that the sequence of MbMAX2 in WT was able to complement the Arabidopsis branching mutant max2,this suggested that the function of MbMAX2 and homologous genes on the model plant was conserved on regulating bud outgrowth.Non-synonymous base mutations in MbMAX2^△sequence of MB were happened,and this leading to the size and site changes of CHAD domain,which was related to signaling transduction.The mutated sequence was unable to complement the branching mutant max2,indicating that the reason of more branching in MB might be resulted from the changed CHAD domain.5.Based on the yeast two-hybrid technology,the interaction between MAX2 and three homologous D14 proteins might be existed.The interacting protein of bait protein MAX2 in apple was screened by using gold yeast two-hybrid system.Ten candidate proteins were preliminarily obtained,which were related to ubiquitination,glucose metabolism,hormone signal transduction,and development.With the sequence of full coding region,however,no protein interactions were identified between MbMAX2 and the candidate proteins in yeast.In conclusion,the function of MAX2 gene on the outgrowth of axillary buds in apple is suppressing by mediating SL signalling pathway.The regulation mechanisms of upstream and downstream of MAX2 in apple,however,need further analyses.