Nucleoside Triphosphate Hydrolases Protein CARD2 Functions In Root Stem Cell Niche Maintenance And Auxin Receptor Protein Stability In Arabidopsis

The phytohormone auxin plays an important part in plant development,such as the initiation and establishment of lateral root primordia,embryogenesis,formation and maintenance of shoot and root apical meristem,leaf vascular tissue development and response to environmental stress.The genes that are activated or repressed during cell division and differentiation are ultimately responsible for the many physiological effects of auxin signal transduction.By this way auxin mediates the different growth and development processes.As the importance of auxin,the aspect of biosynthesis,degradation,transport and signal transduction have been reported widely.The purpose of this study is to find novel gene related to auxin.By screening the DR5::GFP signal in Arabidopsis seeds mutagenized by EMS we obtained constitutive auxin-responsive with DR5::GFP mutant card2.By map-based cloning and sequencing we confirmed the mutation of CARD2 as a conversion of G to A,which caused a premature translation termination.CARD2 is an ortholog of SEN1 in the yeast Saccharomyces cerevisiae,SEN1 functions in many nuclear processes,including transcription termination,DNA repair,and RNA processing.In card2 mutant,the DR5::GFP signal is stronger in root,hypocotyl and leaf vein.The quantity of root meristem cells is more in card2than in WT.CARD2 is mainly expressed in root and rarely in shoot.The subcellula localization of the protein is in nucleus and clustered around the nuclear membrane.The phenotypes of CARD2 overexpression lines are contray to the card2 mutant with weaker DR5::GFP signal and decreased number of root meristem cells.The expression of CARD2 is induced by NAA and heat shock and repressed by cytokinin.CARD2 functions in the maintenance of root stem cell niche.By genetic screening,we found that QC divided more frequently in card2 than in WT,and the trend is the same for cortical/endodermal cells indicated by the pSCR::GFP and pSHR::SHR-GFP markers.The larger expression area of J2341 and Q1630 indicated CARD2 is involved in the extension and differentiation of distal stem cells.All these support that the maintenance of root stem cell niche in card2 mutant is disturbed.The auxin signal is enhanced in card2.By introducing the auxin indicative markers such as PLT1::PLT1-YFP,PLT2::PLT2-YFP and R2D2 in card2,we observed that the auxin signal is stronger in card2 than WT.CARD2 is involved in the pathway of SCF^TIR1-dependent degradation of AUX/IAA proteins.In card2,the level of HS::AXR3NT-GUS is increased.By treating the seedlings carrying HS::AXR3NT-GUS in 37?dark for 2h,followed by 22?light for different time and then measuring GUS activity by 4-methylumbelliferyl-b-D-glucuronide?MUG?assay,we concluded that GUS degraded faster in card2 than in WT.Through GUS staining and western blot we confirmed that mutation in CARD2 increased the stability of auxin receptor protein TIR1.The transcription level of heat shock proteins is increased and there are more changes of heat shock proteins in card2 than WT,which supports that the increased stability of TIR1 is resulted from the increased level of HSPs.We tested that CARD2 interacted with nucleoporins NUP58 by carrying yeast two-hybrid,biomolecular fluorescence complementation in tobacco and colocalization in the cell membrane.card2 plants are deficient in RNA with polyA export.