Mechanism Of Transcription Factors MYB94 And MYB96 In Inhibiting Somatic Cell "Dedifferentiation" In Arabidopsis Thaliana

Plant somatic cells can be reprogrammed during in vitro culture due to their totipotency,and regenerate shoots through "dedifferentiation" and "redifferentiation".Callus induction after somatic cell "dedifferentiation" is the initial step of a typical plant regeneration system.Several callus formation regulators have been identified,such as ARF7.However,the molecular control of callus formation is largely unknown.The MYB transcription factors represent a large family of transcription factors in all eukaryotes.In plants,MYB transcription factors are shown to be involved in the regulation of plant development and responses to biotic and abiotic stresses.However,participation of MYB transcription factors in the regulation of callus formation has not yet been described.In this study,analysis of microarray data showed that two MYB transcription factors,MY1394 and MYB96 were specifically expressed at high levels during callus formation.The function and regulatory mechanisms of them in callus formation were studied and the results were as follows:1.MYB94 and MYB96 inhibited both callus initiation and growthEthynyl deoxyuridine(EdU)staining showed that after 2 d on CIM,myb94,myb966 generated larger numbers of cell clusters than the WT,and myb94 myb96 produced the largest.Tissue clearing showed that the frequencies of outgrowth initiation were higher from root explants of myb94,myb96,and myb94 myb96 than from that of the WT..After 18 days' culture on CIM,myb94,myb96,and myb94 myb96 generated more and larger calli than the WT,with myh94 myb96 producing the most.MYB94 and MYB96 were expressed in the newly formed callus.The results show that MYB94 and MYB96 redundantly inhibit both callus initiation and growth.2.MYB94 and MYB96 inhibit callus initiation via directly repressing the transcription of LBD29qRT-PCR and GUS staining showed that LBD29 expression was increased in myb94,myb96,and myb94 myb96 background compared to that in Col-0.EMSA and ChIP anslysis showed that MYB94 and MYB96 could directly bind to the promoter of LBD29.Mutation of LBD29 in myb94 myb96 rescued its callus initiation phenotype,but not callus growth phenotype,suggesting that MYB94 and MYB96 act upstream of LBD29 during callus initiation,but not during callus growth.The above results show that MYB94 and MYB96 inhibit callus initiation via directly repressing the transcription of LBD29.3.MYB94 and MYB96 might inhibit callus growth via regulating VLCFA biosynthesisCompared to Col-0,the expression of KCS1,KAS1 and PAS1 were decreased in myb94 myb96.Thus,MYB94 and MYB96 might inhibit callus growth via regulating VLCFA biosynthesis.4.ConclusionsMYB94 and MYB96 are up-regulated during CIM incubation and expressed in newly formed callus.They play inhibitory roles in callus initiation via directly repressing the transcription of LBD29 and also inhibit callus growth via regulating VLCFA biosynthesis.