Exploring The Role Of SEUSS In Arabidopsis Root Development

Arabidopsis is a model dicotyledonous plant,because of its small genome,self pollination,little overall genetic variation,and various mutant materials through genetic engineering more easily and rapidly than any other plant genome mutants.So it is a good material for genetic research.Arabidopsis root structure is simple and was divided into three zones,including the meristem zone,elongation zone and the differentiation zone.Arabidopsis root is a closed type radial structure,from outside to inside is epidermis,cortex,endodermis and stele.As the root age,middle cortex(MC)will occur randomly in the endodermis.The mechanism of MC formation is complex and needs many regulators,such as SCR,SHR,SCL3,and the regulation of hormones,such as auxin,GA.In order to explore the role of SEUSS in Arabidopsis root development,we observed the root phenotype of seu mutant,and found that loss of SEUSS function led to larger root meristem size and MC formation.The results of this paper were as followings:1.Primary root of seu mutant was longer than wt because of its more cell division in meristem zone.2.Expression pattern of pSEU::GUS/GFP and pSEU::SEU:GFP were widely expressed in the root tip.Transcript expression of SEUSS was induced by auxin,inhibited by GA and induced by PAC.3.QC46,WOX5::GUS,pPLT2::PLT2:EGFP,pPLT3::ERCFP in seu were down-regulated expression.Expression of SCR and SHR in QC of seu were decreased.These results show SEUSS positively regulate the QC and the stem cell niche activity.4.Expression levels of DR5::GUS and DR5::GFP in seu were lower than in wt,especially in QC.Lower auxin content in QC may explain QC activity in seu.Expression of PIN1,PIN2,PIN3,PIN4 in seu were decreased,led to auxin content in seu decreased.The sensitivity of seu mutants to exogenous auxin was weaker than that of wild type,these results showed SEUSS plays some role in auxin signaling pathway.5.Compared with WT,MC formation in seu was earlier and has a higher frequency.Consistently,overexpression of SEUSS by the 35S promoter(35S::SEU)markedly inhibited MC formation.Expression of a SEU-GFP fusion driven by the SCR promoter(SCR::SEU-GFP)in WT could mimic the inhibitory effects of 35S::SEU,whereas SCR promoter-driven expression of SEU-RNAi(SCR::SEU-RNAi)in WT was able to increase the frequency of MC formation,suggesting that SEUSS controls the timing and extent of MC formation in a cell-autonomous manner.6.Expression of pSCR::H2BYFP,pSHR::H2BYFP,pSHR::SHR:GFP,pSCL3::GUS,pSCL3::SCL3;GFP were decreased,suggesting that SEUSS is an ustream activator of SHR,SCR and SCL3.In seu-4/shr-2 double mutants,a single ground tissue layer was observed,as seen in shr-2.This finding is consistent with previous reports showing that SHR is indispensable for MC formation.The seu/scr-3 double mutants displayed a significantly higher frequency of MC formation than either of the single mutants,scr-3 and seu,suggesting SCR and SEUSS were involved in controlling the MC formation.By contrast,the MC formation phenotype in seu-3/scl3 was similar to that in seu-3,indicating an epistatic relationship of SCL3 to SEUSS in the control of MC formation.These results showed SEUSS has distinct genetic interactions with SHR,SCR and SCL3.Yeast one-hybrid and ChIP-qPCR assays indicated that SEUSS could bind to the promoter regions of SCR,SHR,SCL3.Thus,SEUSS may act directly on SCR,SHR,SCL3 to inhibit the formation of MC.7.The rbr-RNAi/seu,jkd-4/seu double mutants displayed a significantly higher frequency of MC formation than either of the single mutants,rbr-RNAi,jkd-4 and seu.These results showed SEUSS,RBR,JKD were involved in controlling MC formation.8.Like as SCL3,SEUSS expression was significantly reduced by GA3,whereas GA deficiency,induced by either PAC or the loss of GA1 function,resulted in an increased expression of SEUSS in the root.However,GA3 was no longer able to suppress the expression of SCL3 in seu-3.In the presence of PAC,the level of SCL3 induction was also attenuated in seu-3.These findings suggest that GA-mediated regulation of SCL3 expression is subject to SEUSS function.Moreover,we found that expression of DELLA proteins were transcriptionally down-regulated in seu-3 but up-regulated in 35S::SEU,indicating that SEUSS mediates the regulation of SCL3 expression by GA signaling.