Rice Lateral Root Specific Marker Lines Collection And Functional Analysis Of Shoebox Gene In Rice Root Development

Root is an important hidden part of the plant.It is important to study the mechanism of root development which was closely related with agronomic traits.In our study,we collected several lateral root specific marker lines by screening enhancer trap lines.Furthermore,we identified and cloned SHB gene by screening T-DNA insertion mutant database,which mediates primary root meristem cell elongation through regulating GA biosynthesis gene KS1,and the reason for shorter cell size in root meristem was analysis in-depth.1.We collected 300 lines for GUS reporter gene expression in lateral root from 15000 rice T-DNA insertion lines.58 of the 300 lines were of genetics stability,and could be act as marker lines for rice lateral root.Besides,we identified several root mutants through screening enhancer trap,and isolated the flanking sequence tags of these mutants.2.shb mutant root phenotype identified.In a rice enhancer trap screen,we isolated a recessive mutant with a short primary root which we have named shoebox {shb).Analysis of median longitudinal sections of root apices of 4-day-old wild-type(WT)and shb seedlings showed that the root meristem size and cell size in meristem of shb was shorter than that of the WT.3.Flanking sequence analysis shows T-DNA was inserted into the fifth introns of LOC_Os05g32270 which encoded an ERF/AP2 transcription factor,and the mutant phenotype was co-segregation with the T-DNA.A genomic fragment containing the LOC_Os05g32270 gene and its promoter could fully complement the mutant phenotypes of shb.We thus concluded that LOC_Os05g32270 is the SHB gene.4.The aerial part of shb mutant plants has typical characteristics of rice GA-deficient or insensitive mutants,such as dark leaves,dwarfism and short internode length.GA3 could rescue the phenotype of short root,small meristem size and short cortiacal cell length in shb mutants,and the levels of bioactive GAs reduced indicated that shb is a novel GA-deficient mutant.5.Microarray data shows the transcription level of many GA biosynthesis genes and transcript factors related with cell elongation in shb mutant changed significantly compared with WT.The results of qPCR analysis of transcript levels of GA biosynthetic genes in wild type shb mutant roots showed KS1 and GA20OX2/SD1 were significantly down-regulated by the shb mutation.EMSA and ChIP-PCR results indicate that SHB can directly bind to GCCGCC motifs in the promoter region of KS1 in vitro and in vivo.SHB encodes an AP2/ERF transcription factor that directly activates transcription of the GA biosynthesis gene KS1.6.RNA in situ hybridization revealed that KS1 is expressed in an overlapping domain with SHB in the root meristem,in agreement with our finding that KS1 is a direct downstream target of SHB.ks1 mutant with severe GA deficiency was found to have smaller root meristem than the WT control.Quantification of cortical cell number and size in the ks1 root meristem revealed that there was a significant decrease in cell proliferation compared to the WT.GA3 could largely restore the number of cortical cells in the h1 root meristem and endogenous GA largely decreased in ks1,suggesting that severe GA deficiency in ks1 is the underlying cause.7.Rice seedlings treated with different concentrations of PAC suggest that GA has a dose-dependent effect on the root meristem development.low concentrations of PAC(1 ?M PAC,like shb)markedly reduced elongation of cortical cells in the WT root meristem without affecting the rate of cell proliferation;high concentrations of PAC(10 ?M,50 ?M,like ks1)could significantly impair cell proliferation in the root meristem,resulting in a smaller root meristem with longer cells.The GA dose-dependent effect on the root meristem development can be found in Arabidopsis.8.SHB functions a positive regulator of GA signaling,and SHB and KS1 are involved in microtubule orientation in proliferating cortical cells.