Mechanism Study Of Arabidopsis AtTLC1 In Regulating Root Stem Cell Niche Maintenance And DNA Damage Response

Plant root growth and development depend on the persistent activity of meristems.The well-organized root meristem of Arabidopsis thaliana contains 4 mitotically inactive central cells,known as the quiescent center(QC),surrounded by different types of stem cells to form the stem cell niche(SCN).The QC cells form an organizing center to maintain the root stem cells.Two main parallel pathways involved in the regulation of specification of the root stem cell niche have been identified: the auxin-PLETHORA(PLT)pathway and the SHORT ROOT(SHR)/SCARECROW(SCR)pathway.During the life cycle of a plant,its root cells are continuously exposed to stresses that can lead to DNA damage.In order to ensure survival and life cyle,organisms have evolved elaborate mechanisms to repair DNA damage.Arabidopsis SCN shows high sensitivity to DNA damage.In order to safeguard genome integrity at the whole-organism level,programmed cell death(PCD)is triggered selectively in root stem cells when suffering from relatively low levels of DNA damage.Sphingolipids are primary endomembrane components in plants and other eukaryotes.Current research showed that sphingolipids participate many biological processes,including signal transduction,programed cell death,vesicalur transport,growth development,response to biological and abiotic stress.Ceramides are the simplest sphingolipids,synthesized by ceramide synthase.Three ceramide synthases have been identified in Arabidopsis,LOH,LOH2 and LOH3,which contain TRAM–Lag1p–CLN8(TLC)domain.The TLC containing proteins may participate in the process of sphingolipids synthesis,while functions of most TLC-containing proteins remain unknown in Arabidopsis.In order to investigate their functions,we screened the corresponding T-DNA insertion lines.Here,we identified a mutant with short primary root(designated attlc1),showing defective maintenance of the root SCN and cell death in the stele stem cells(SSCs).The major results obtained are as follows: 1)Mutation of AtTLC1 causes defects in QC identity and differentiated columella stem cells,which leads to the defective cellular organization of the root SCN,small size of root meristem and primary root length.2)IAA measurement and auxin-responsive marker DR5:GFP and DR5:GUS indicate that AtTLC1 mutation primarily affects auxin distribution by reducing the expression level of several PIN genes and AtTLC1 expression is induced by auxin.3)Loss of AtTLC1 affects PLT1 and PLT2 expression,while has no effect on SHR/SCR,indicating that AtTLC1 participates in the regulation of root SCN through the auxin/PLT pathway.4)Further assays indicate that PLT2 induces the expression of AtTLC1 and there may exit a positive fed-back between AtTLC1 and PLT1/2 in maintaining root SCN patterning.5)AtTLC1,which contains a TLC domain,localizes on both ER and Golgi to perform its function.Quantitative sphingolipids profiling showed that attlc1 has lower level of sphingolipids,especially in Glycosyl inositolphosphoceramides(GIPCs)with very long chain fatty acids,suggesting AtTLC1 is responsible for the homeostasis of sphingolipids in Arabidopsis.6)The ceramide synthase inhibitor Fumonisin B1(FB1),which can disturb the homeostasis of sphingolipids,represses the expression of SCN regulators PLTs,and attlc1 mutant shows high sensitivity to FB1,suggesting that AtTLC1 may regulates root SCN by maintaining the sphingolipids homeostasis.At the same time,attlc1 mutant showing cell death in the SSCs is hypersensitive to agents that induce double-stranded DNA breaks and has an increased expression of DNA damage response genes,indicating that the DDR system is activated in the attlc1 mutant.Cell death phenotype disappears in attlc1sog1-1 double mutant,suggesting that SOG1 mutation suppresses the activated DDR system in attlc1,thus indicaing that AtTLC1 acts through DDR pathway in response to DNA damage.ERF115 is activated by DNA damage in attlc1 root tips,inducing QC division,thus replenishing the dead stem cells.In this paper,through the molecular mechanism of the mutant,we have a more in-depth understanding of the mechanism of root stem cell maintenance.