Regulation Of The Meristem Activity By Reversibly Glycosylated Polypeptides RGP1 And RGP2 In Arabidopsis

In higher plants,organ formation occurs continuously and repetitively from apical meristems at their growing tips.This plasticity enables plants to adapt their body shape in response to developmental,physical and environmental cues.The shoot apical meristem(SAM)originates during embryogenesis and later initiates the aerial part of higher plants such as the stem,leaf and flower.Within the SAM,a small number of unspecialized immature stem cells are confined to the upper region of the central zone(CZ),serving as the ultimate source of all shoot cells.WUSCHEL(WUS),a homeodomain transcription factor expressed in the organizing center(OC)of the Arabidopsis SAM,is a key regulator controlling SAM stem cell populations.However,the molecular mechanisms underlying WUS function in stem cells are still challenging.Previously,we identified the factors that cooperate with WUS in the SAM,including Reversibly Glycosylated Polypeptides 1(RGP1)and RGP2.Here,we demonstrate that RGP1 and RGP2 act as direct interacting cofactors of WUS.Furthermore,RGP1 and RGP2 participate in the regulation of root apical meristem activity.Although RGP1 and RGP2 have been proved to function in the polysaccharide biosynthesis of plant cell walls,their roles in the regulation of stem cell activity in SAM remain unknown.Here,we analyzed their functions in meristem maintenance.The main results are as follows:(1)We confirmed the direct interactions between WUS and RGP1 or RGP2 by in vivo Co-immunoprecipitation(Co-IP)assays and in vitro pull-down assays,respectively.Therefore,RGP1 and RGP2 proteins function as WUS-interacting cofactors in SAM;(2)RGP1 and RGP2 were detected to be widely expressed in shoot and inflorescence meristems,as well as the younger leaves.In the SAM,RGP1 was strongly expressed in the OC,overlapped with WUS expression region.RGP2 was expressed in the whole SAM;(3)The expression of both RGP1 and RGP2 was suppressed in the hpRGP1/2 line,which harbouring a hairpin(hp)construct specifically targeting RGP1 and RGP2 transcripts.The sizes of shoot and inflorescence meristems were smaller in hpRGP1/2 compared with the wild type.The expression of SHOOT MERISTEMLESS(STM)and CLAVATA 3(CLV3)were both reduced in the SAM of hpRGP1/2 line compared with the wild type,while the expression pattern of WUS-GFP3 was almost not changed.The expression levels of the WUS-target genes,such as CLV3,TOPLESS(TPL)and TONOPLAST INTRINSIC PROTEIN 2(TIP2),were decreased in the hpRGP1/2 line compared with the wild type,indicating that RGP1 and RGP2 regulate the expression of the target genes of WUS through directly interacting with WUS in the SAM.(4)RGP1 and RGP2 were expressed in the whole root.Stronger expression signals were detected in the root tips.The roots of the hpRGP1/2 line were obviously shorter than those of the wild type,with abnormal cell division in the quiescent center.The expression of RGP1 and RGP2 were also detected in embryo sac and embryos.The embryogenesis of hpRGP1/2 was defective.These results suggest that RGP1 and RGP2 are involved in various developmental processes,including embryogenesis and post-embryonic shoot and root development.