Molecular Mechanism Of Root Development By Transcription Factor NAC1 In Arabidopsis

Root not only grants plants with a more efficient foraging of water and nutrients,but also provides anchorage to allow growth in height.Also,roots have the function of synthesizing organic substances and conducing nutritional reproduction.The RAM(root apical meristem),is consisted of undifferentiated cells that give rise to the different cells of the root.In the model plant Arabidopsis thaliana,the primary root RAM is composed of four sets of initial stem cells surrounding with a group of mitotically less active QC(quiescent center)cells.This regular pattern is established and maintained by different sets of stem cells residing in the root meristem.QC cells and the actively dividing stem cells form a specialized cellular microenvironment.The PLT(PLETHORA)and SCR/SHR(SCARECROW/SHORTROOT)transcription factors,which are required for QC specification,are involved in both stem cell maintenance and the differentiation of their progeny.The GRAS family transcription factors SCR and SHR,act as a heterodimer,play a critical role in ACD(asymmetric cell division)of CEI(cortex/endodermis initial)and CEID(cortex/endodermis initial daughter),and are required for the specification and maintenance of the root stem cell niche.SHR moves from internal tissue to the endodermis and further movement is restricted by SCR.SCR and SHR directly activate the D-type cyclin CYCD6;1,whose expression is enriched in the CEI/CEID cells and induced extra ACDs.In cycd6;1 mutant seedling,CEID cells showed significantly fewer formative divisions.By 7-14 days post-germination,the endodermis asynchronously undergoes an additional periclinal ACD,resulting in the production of an additional cortex layer,named as middle cortex.Therefore,middle cortex formation is considered to be an indicator of root ground tissue maturation.Taken together,SCR/SHR-CYCD6;1 plays an essential role in regulating ground tissue formation.NAC(NAM/ATAF1,2/CUC2)-domain proteins comprise a large,plant-specific of transcription factors involved in many aspects of plants development.During lateral root initiation,NAC1 acts as a transcription activator to mediate auxin signaling.Based on the GUS staining analysis with a translational fusion to GUS reporter line(NAC1pro:gNAC1-GUS),a high expression of NAC1 was observed in root meristem and ground tissue.We obtained two T-DNA insertion NAC1 mutant lines,which showed reduced root length and an increased frequency of plants exhibiting root middle cortex.However,the proportion of plants exhibiting root middle cortex was greatly reduced in NAC1 overexpression lines.According to cytological analysis,the size of the RAM and meristem cell number were reduced in the nac1 mutants compared to wild type plants.Furthermore,the results showed that NAC1 directly binds and inhibits the transcription of the G1/S cell cycle regulated gene E2Fa(E2promoter-bindingfactor a).Loss of NAC1 triggers an up-regulation of the E2Fa expression,causing a reduced meristem size and short-root phenotype which are largely rescued by mutation of E2Fa.Furthermore,NAC1 recruits the co-repressor TPL to maintain root growth.This study provides evidence to show that NAC1 together with TPL maintains root meristem size and root growth by directly repressing the transcription of E2Fa.Given the important role of a key component of the cell cycle regulator CYCD6;1(CYCLIND6;1)in root middle cortex formation,we examined the effect of NAC1 on the expression of transcription reporter CYCD6;1pro:GFP and found that an increased GFP fluorescence in the endodermis in the nac1 mutants.In the cycd6;1 mutant seedling,the endodermis showed significantly fewer periclinal cell divisions.The excessed periclinal cell divisions in the endodermis in the nac1-1 mutant was largely rescued in the nacl-1/cycd6;1 double mutant.These results indicated that NAC1-CYCD6;1 regulatory module controls root endodermal cell division to limit production of middle cortex cells.Furthermore,we performed chromatin immunoprecipitation(ChIP)-qPCR and electrophoretic mobility shift assay(EMSA),and found that NAC1 directly inhibits the expression of CYCD6;1 through recruiting TPL.Next,we examined three tpl mutants(tpl-1,tpl-9 and tpl-10)and found that all these three mutant alleles showed an increased frequency of plants exhibiting root middle cortex as the nacl mutants.In contrast to the CYCD6;1pro:GFP expression in the middle cortex in wild type roots,an increased proportion of roots with expanded CYCD6;1pro:GFP expression and an enhanced GFP fluorescence in the endodermis were observed in tpl mutant Collectively,our results showed that NAC1 recruits TPL to inhibit CYCD6;1 expression and excessive periclinal cell divisions of the endodermis during root ground tissue maturation.Proper patterning of the root ground tissues requires the SCR/SHR transcription factor complex.Yeast two-hybrid(Y2H),Co-IP,BiFc and split luciferase complementation assays showed that NAC1 interacts directly with SCR and SHR in vitro and in vivo.To gain insight into the genetic relationship between NAC1 and SCR or SHR,we generated the nacl-1/scr-2 and nacl-1/shr-2 double mutants and examined the root middle cortex formation phenotypes.The results showed that,the double mutants were similar to that observed in the scr-2 or shr-2 single mutant,respectively.We further examined the effects of NAC1,SHR and SCR on the local transcription of CYCD6;1(CYCD6;1pro:GFP),and found that the GFP expression was largely up-regulated in the endodermis of nacl-1,scr-2,and nac1-1/scr-2 compared with of the wild type,whereas the expression of CYCD6;1 almost disappeared in shr-2 or nacl-1/shr-2.Taken together,these results indicated that both SHR and SCR are genetically epistatic to NAC1 and required for NAC1 to repress root middle cortex formation.Additionally,NAC1 and SCR directly interact and form an interdependent module to repress the expression of CYCD6;1.However,NAC1 and SHR repress each other to affect the DNA-binding activity of CYCD6;1.Our study provides mechanistic insights into how the NAC1-TPL module integrates with the master transcriptional regulators SCR and SHR to control root ground tissue patterning by finetuning the spatiotemporal expression of CYCD6;1 in Arabidopsis.This study analyzed the biological regulation of NAC1 in the primary root growth and middle cortex formation in Arabidopsis.This study not only deepened our understanding of the complex functions and important regulatory role of NAC family,but also provided important clues to clarify the molecular mechanism of plant root meristem development and stem cell ACDs.