Mechanism Analysis Of RSC1 Regulating The Root Stem Cell Niche

The root stem cell niche(SCN)plays a major role in the regulation of root morphogenesis and development.This special structure is composed of quiescent center(QC)and surrounding stem cells.Current researchs find that the root SCN is regulated by several transcription factors including WOX5,SCR/SHR,PLT1/PLT2 and various hormones including auxin,cytokinin,ABA.ethylene,GA and so on.However,how do these transcription factors maintain the root SCN?Are they synergistic or antagonistic?Is there a signal interaction between different plant hormones in this process?There Is insufficient evidence to answer the these questions.In order to further investigate the regulatory eechanisn of root stem cells,we focused on the search for new stem cell regulatory factors.In this research,mutants with defective SCN were screened on purpose.Therefore,the rseI(root sten cell 1)mutant was obtained.In the present study,the molecular mechanism by which RSC1 controls root SCN identity was investigated using genetics,molecular biology,cell biology and other techniques.First,we find that the rscl-1 mutant is caused by a n,utation in the gene encoding adenosine three phosphate hydrolase.We generated pRSC1::GUS and pRSC1::GFP-RSC1 transgenetic lines to stLidy the RSC1 expression pattern and the RSC1 protein sub-cellular localization.Analysis of several pRSC1::GUS and pRSC1::GFP-RSC1 transgenic lines revealed that RSC1 was highly expressed in the root SCN.indicating that RSC1 might control the SCN.In addition,pRSC1::GFP-RSC1 could recovery the phenotype of rscl-1.Fluorescent staining and western blot showed that the RSC1 protein was located in mitochondria.Previous study showed that mitochondria are the major intracellular organelle that produce ROS(Reactive oxygen species).Morever,the level of ROS plays an important role in SCN development.It seems that RSC1 might control the root SCN through regulating the homeostasis of ROS.To prove the hypothesis,we detected the level of ROS in rscl.Compared with wild type Col·0,the level of ROS was decreased in rsc1 and increased in 35S::RSC1.Furthermore,treatment of rsc1 with MV could decreased the high QC division and DSC differentiation rate,suggesting that the phenotype of rsc1 was caused by the decreased ROS level.The regulation mechanism of ROS in plants was complex.To further explore the mechanisms of decreased ROS level in rsc1 mutant,we evaluated the expression of ROS-related genes.The qRT?PCR results showed that the expression of APX2 which encoding ROS-scavenging enzyme was up-regulated in rscl.Additionally.resluts of yeast two hybrid confirmed that RSC1 intracts with CBSX3,CBSX3 is located in mitochondria and influence the ROS level through controlling the Trx of NADP-Trx system.Consequently,RSC1 regulated the level of ROS through control the exoression of APX2 and the interaction with CBSX3.In order to investigating the downstream regulatory factors of RSC1,we detected the expression of WOX5,PLT1/PLT2,SCR/SHR in rsc1 through qRT?PCR and confocal.The expression of SCR/SHR was down-regulated in rsc1,while expression of WOX5,PLT1/PLT2 did not show significant difference between rsc1 and the WT control.These results indicated that SHR and SCR are involved in RSC1 regulated SCN maintenance.Meanwhile,we found that the overexpression WOX5 could depressed the expression of RSC1,indicating that WOX5 might control RSC1.In addition,the signal of DR5rev::GFP had no variety between rsc1 and WT.and the expression modle of RSC1 was not affected by various hormones.These results illustrated that RSC1 might regulate QC cell division and root DSC identity independent of plant hormones.In conclusion.our research discovered a novel regulatory factor of SCN named RSC1 and specifically expressed in root tip.Consequently.RSC1 regulate the level of ROS through controlling the expression of APX2 and interacting with CBSX3.thus regulating the expression of SCR and SHR,involved in root SCN maintenance eventually.