The Function Of Topoisomerase ?? On Arabidopsis Root Stem Cell Development

Topoisomerase I(TOP1)is an important nuclear enzyme involved in many aspects of DNA metabolism,such as DNA replication,RNA transcription and performs its topological transformation reactions on DNA via a concerted breakage/religation mechanism.The plant source anti-cancer alkaloid camptothecin(CPT)targets TOP1 to block religation of the normally transient TOP1-DNA cleavage complex(TOPlcc)to result in DNA damage induced cell death and cell cycle arrest in a intercalation manner.These are two TOP1 paralogs(TOP1? and TOP1?)in Arabidopsis genome.top1? results in a delayed progression of meristem cells into differentiating organ primordia,larger meristem,fasciations of the inflorescence stem in aboveground and cell death in root stem cell niche,however,top1? has no obvious phenotype.The diffference between the two genes is largely unknown.The mechanism of the root stem cell death in top1? and the expression patterns of them are need to be elucidated.CPT target in Arabidopsis is still indefinite.Here,the main results as follows:1.We construct the promoter and protein reporter lines of TOP1? and TOP1?.The expression patterns of the two genes were largely identical.TOP1? protein fusion could functionally complement top1? mutant phenotypes.These indicated that the two protein are functional redundant and also distinct.2.Reduced root meristem of top1? mutant shorten root elongation.3.Exogenous CPT induced same root phenotypes as top1? mutant.CPT introduced DNA damage all through the root meristem and stem cell niche.CPT induced G2 phase cytotoxicity and S phase dependent cell death in meristem and stem cell niche,separately.The meristem cells responsed to CPT cytotoxicity in ATM/ATR/WEE dependent.In contrast,top1? mutant induced DNA damage and S phase related cell death in limited stem cells.a series of cell cycle regulation genes(CYCB1;1,WEEl et al.)changed upon CPT time course treatment.All these evidence conclude CPT introduces S phase related cell death and G2 arrest.4.The site mutation of TOP1? in conserved CPT resistant site,N871S,resulted strong CPT resistant in Arabidopsis.Combined with results of phenotypes and changed cell cycle genes,data show TOP1? is the in vivo target of CPT.5.Both CPT and topla mutant could induce ERF115 and WOX5 up-regulation.ERF115 expression domain was in cells around the dead stem cells.The ERF115 arised followed by the appearance of dead cells,and before up-regulation of WOX5.These indicated both CPT and top1? mutant induce sternness to replenish the dead stem cells and maintain stem cell niche.6.CPT up-regulates ERF115 and WOX5 through inhibition of TOP1? induction of elevated H3K27me3 histone modification level.7.Histone H3K27 demethylase inhibitor GSK-J4 repressed CPT induced up-regulation of ERF115 and WOX5.The evidence suggested the mechanism of epigenetic modification of H3K27me3 in regulation of stemness relative genes.8.GSK-J4 decreased root recovery from CPT cytotoxicity which elucidated a way of combinatorial treatment of GSK-J4 and CPT to clean the potential sternness.