Mechanism Research Of Arabidopsis BHLH Transcription Factor HBI1 In Callus Initiation

In vitro organogenesis is a process that in vitro tissues or a mass of cells(callus)differentiate into new organs,such as in vitro shoot,root or flower.An efficient shoot regeneration protocol in Arabidopsis includes two steps:callus formation and shoot regeneration.Meanwhile,the first mitosis of somatic cells(dedifferentiation)is an initial step for callus formation as well as a key step for shoot regeneration.Therefore,carrying out the research about molecular mechanisms of callus initiation has great significance for comprehensive analysis of totipotency of plant cells and the regulatory mechanisms of shoot regeneration.We previously identified an auxin response factors ARF10,which promoted callus initiation.Based on this discovery,we obtain a bHLH protein named HOMOLOG OF BEE2 INTERACTING WITH IBH1(HBI1)which could interact with ARF10.In this article,we demonstrate that HBI1 promotes callus initiation and formation through directly up-regulating the expression of WOX12,TIR1 and AXR2.Furthermore,we show that ARF10 directly activates WOX12 transcription and HBI1 and ARF10 act synergistically in regulating the transcription of WOX12.Besides,we also find that miR393a inhibits callus formation and shoot regeneration via repressing TIR1.The main research process and results are as follows:1 Molecular mechanism of HBI1 promotes callus initiation from in vitro culture.1.1 HBI1 promotes callus initiation and formation from in vitro cultured root explantsVia yeast two hybrid and BIFC,we obtained a bHLH protein,HBI1,that could physically interact with ARF10.We tested the callus formation ability of root explants from both gain-of-function and loss-of-function HBI1 plants.The results indicated that HBI1 played a crucial role in callus formation.Ethynyl deoxyuridine(EdU)staining tested the influence of HBI1 on cell proliferation during callus initiation.The results indicated that HBI1 promoted the transition from G1 to S stage of cell cycle progression during callus initiation.Compared the difference of pCYCB1;1 expression in HBI1-OX and HBI1-SRDX background.The results indicated that HBI1 accelerated the rate of proliferation during callus initiation.1.2 HBI1 promoted the expression of WOX5,WOX12,TIR1 and AXR2 during callus formationThe gene expression test showed that the expression levels of WOX5,WOX12,TIR1 and AXR2 were up-regulated in HBI1-OX,while down-regulated in HBI1-SRDX when compared with the Col-0.Compared the difference of WOX5 expression in HBI1-OX and HBI1-SRDX background.The results indicated that HBI1 possibly accelerated the cell fate transition from founder cells to root meristem like callus by promoting the expression of WOX5.Through EMSA,ChIP and LUC experiments we demonstrated that HBI could directly bind to the promoter of WOX12 to activate its expression.The phenotype of callus formation analysis indicated that WOX12 promoted callus formation.EdU staining suggested that WOX12 promoted the transition from G1 to S stage of cell cycle progression during callus initiation.Genetic complementation test confirmed that WOX12 acted downstream of HBI1.Through EMSA and ChIP experiments we showed that HBI could directly up-regulate the expression of TIR1.Identification of callus formation ability suggesting that TIR1 promoted callus formation.Genetic complementation test showed confirming that TIR1 acted downstream of HBI 1.We also demonstrated that HBI1 was able to directly activate the transcription of AXR2.The phenotype analysis suggesting that AXR2 promoted callus formation.Via EMSA and LUC experiments we demonstrated that ARF10 directly activated WOX12 transcription.Furthermore,LUC activity detection showed that transiently expressed HBI1 or ARF10 resulted in a significant increase of pWOX12-driven LUC activity when compared to the control effector,and co-expression of HBI1 and ARF10 could enhance such activity,supporting that HBI1 and ARF10 acted synergistically to activate WOX12 transcription.To sum up,through studying the function of HBI1,an interacting protein of ARF10,we found a new regulatory mechanism that HBI1 promoted callus initiation.Our works laid a good foundation on the molecular mechanism exploration of plant regeneration.2 miR393a inhibits callus formation and shoot regeneration in Arabidopsis thaliana via repressing TIR1The phenotype analysis of Col-0,p35S::miR393a-1,p35S::miR393a-2,p35S:MIM393-1 and p35S:MIM393-2 explants indicated that miR393a inhibited callus formation and shoot regeneration in in vitro culture.Gene expression difference analysis of WUS and CLV3 indicated that miR393a had a negative effect on the de novo formation of SAMs from in vitro culture.The expression of DR5 was examined and suggested that miR393a inhibited the auxin response during shoot regeneration in in vitro culture.As a target of miR393a,the phenotype of TIR1 related mutants was analyzed.The results showed that,comparing with the Col-0 explants,the callus formation and shoot regeneration ability was enhanced in pTIR1::mTIR1-1 and pTIR1::mTIR1-2 explants.Moreover,pTIR1:mTIR1-2 could rescue the p35S:miR393a-2 phenotype.All these results demonstrated that miR393a inhibited callus formation and shoot regeneration via repressing TIR1 in Arabidopsis thalianaIn conclusion,through studying the function of miR393 and TIR1,our study provides new findings of the molecular mechanisms that the miR393-TIR1 molecular regulation pathway control callus formation and shoot regeneration in in vitro culture.