Intact CTD Of RNA Polymerse ? Maintains The Root Stem Cell Niches Mediated By Cell Cycling Control In Arabidopsis

The full length of RNA Pol ? C-terminal domain CTD plays a critical role in the development of plants and animals.An EMS mutant card1-1(constitutive auxin response with DR5::GFP)was identified in which there is a mutation in the border of the 11 th exon of RPB1 and encodes a truncated protein with shortened CTD tail.Phosphorylation of the CTD repeats of RNA polymerase ?'s largest subunit RPB1 is coupled to transcription,pre-m RNA process and DNA replication in many eukaryotes.The truncated CTD of RPB1 in card1-1 disturbed cell cycling and enlarged shoot and root apical meristem size.The root stem cell niche marker data demonstrated that full length of CTD of RPB1 is necessary to define the distinctive exprsssion of genes responsibility for cell fate determination.CARD1 also participated in auxin signaling and maintenance of root stem cell niche.Investigations into the importance of CTD length,in yeast and mammals,have been widely studied.This is the first time,to our knowledge,demonstrating the developmental role of CTD in plants in vivo.Our present studies will extend the knowledge of RNA Pol ? in developmental process of life.(1)CARD1 is RPB1 and the largest subunit of RNA Pol ?.We mutagenized seeds carrying the auxin-responsive reporter DR5::GFP with EMS,then we obtained the mutant of card1-1.We confirmed that card1-1 is RPB1 through mapping.The genomic DNA of RPB1 is 7472 bp,encodes 1839 amino acid.The C-terminal domain(CTD)of RPB1 consists of multiple heptad repeats(consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7).In Arabidopsis,39 repeats of the consensus sequence are present in RNAP ? CTD.The mutation site of card1-1 is the 31 th repeat just in the border of the 11 th exon of RPB1 and encodes a truncated protein with shorten CTD tail.(2)CARD1 protein restricted the shoot and root meristem cell size.All lateral organs,including leaves,flowers and petals,are produced from SAM,IAM,and FM.The shoot and root meristem cell was enlarged in the card1-1 mutant.The activity of Pro CLV3:GUS in the card1-1 mutant had leakage-expression in other places in the SAM.Pro WUS:GUS expression domain in card1-1 was biger than Col-0.The root tip of card1-1 showed larger meristem with smaller cortex cells than Col-0.It indicates that CARD1 protein has a function in the maintainence of apical meristem activity.(3)CTD domain truncated accerelated cell cycling.The number of meristem cell increased in card1-1 mutant,Pro CYCB1;1:GUS expression indicated the meristem cell of card1-1 is more activity.Cell-cycle progression requires the sequential association of the CDKs with different types of cyclin.Cell-cycle checkpoint protein is diffferently up regulated in card1-1 mutant,such as S phase protein which DNA synthesis,CYCB family which regulates G2-M transition and CYCD family which regulates G1-S transition.Flow cytometry analyses indicated that the DNA contents of 2C+4C+8C were increased in card1-1 mutant,the endoreplication is also decreased.It indicated that card1-1 accelerated cell cycling.(4)CARD1 functioned in the maintenance of root stem cell niche.A critical issue in development is the coordination of the activity of stem cell niches with differentiation of their progeny to ensure coherent organ growth.QC divided frequently in card1-1 mutant,J2341 and Q1630 indicated that card1-1 accounted for the extention of CSC and distal stem cell differentiation.Pro CYCD6;1:GUS also indicated that CEI/CEID expression increased in card1-1 mutant.We also found that the cortex and endodermis divided frequently.(5)RNA-seq analysis showed that the transcription activity were more activity in card1-1 mutant.Go term showed that genes response to endogenous stimulus and root development gene were clustered.Q-PCR analysis indicated that Cell cycling gene and RNA complex activity were up regulated,we also found that the induction of heat shock gene were up regulated.HS::AXR3NTGUS indicated that the induction of transcription level were also up regulated.(6)Full length CTD of RPB1 is required to maintain its' stability and phosphorylation status.CTD phosphorylation is affected by its length and composition.We created serious of CTD domain truncated card1 s mutants which demonstrated that the importance of the full length of CTD to plant development.Phosphorylation of the CTD functioned in transcription progress,the phosphorylation level of CTD was altered in card1 s mutant.(7)Phosphorylation of the CTD is an important process for transcription-coupled events in yeast and mammals.The transcription pre-initiation complex recruits hypophosphorylated RNA polymerase ?(RNAP ?)to the promoter,now the CTD Ser5 P is high.After promoter release,the level of the CTD Ser5 P mark gradually decreases owing to the action of CTD phosphatases,Whereas Ser2 P levels increases through the CDKDs.Ser5 P level is decreased in card1-1 mutant.Moreover both of card1-1 and cpl3-1 display the higher ratios between S2 P and S5 P than wild type.cpl3-1CYCB1;1:GUS expression also increased and similar with card1-1.So the higher ratios between S2 P and S5 P level might results cell cycling accelerated.(8)CARD1 participated in auxin response but independent of SCFTIR1/AFBs Complex. card1-1 constitutive to auxin response,however,axr1-3card1-1 could compensated the insensitive of axr1-3 to auxin response.So its function might independent of SCFTIR1/AFBs Complex.card1-1 couldn't participate in auxin synthesis and transport.Some of ARFs genes were up regulated also demonstrated that card1-1 not directly participated in auxin signaling.(9)In mammalians,the promoter-proximal pausing of RNA Pol ? is widely studied.Phosphorylation of the CTD is associated with RNAP ? promoter-proximal pausing.In plants,the promoter-proximal pausing is not reported now.CHIP-QPCR analysis showed that Pol ? is enriched in the TSS region of CYCB1;1 gene,but it has a weak bind in card1-1 mutant than WT.So we hypothesized that the low pausing in TSS might results the whole genome transcription up regulated.