Molecular Identification And Analysis Of Novel TTG1 Alleles Modifying Root Hair Pattern Formation In Epidermis Of Arabidopsis Roots

Root is an important organ in the process of plant growth and development.Root hair,as an important part of root,plays a key role in the absorption of water and nutrients.Root hair is formed by epidermal cells of root tip.The development of root epidermis of Arabidopsis is widely used as a simple model to study root hair differentiation and development.Previously studies have shown that there are two distinct cell types produced in the root epidermis,root-hair cells(H cell)and non-hair cells(N cell),and these are arranged in a position-dependent pattern.The pattern of root-hair and non-hair epidermal cells in the Arabidopsis root is governed by a network of transcriptional regulators.The core Myb-b HLH-WD40 transcriptional complex includes the WD40-repeat protein TRANSPARENT TESTA GLABRA1(TTG1).In this study,we indentified a double mutant cpc-1 ttg1-23 by conducting an EMS-based enhancer screen with the cpc-1 GL2::GUS line.ttg1-23 mutation was detected by combining the genetic mapping and whole genome sequencing,then verified and analyzed the function by means of molecular biology technology,which aimed at further analyzing the mechanism of root hair differentiation and development in crop plants.The main results were listed as follows.1.The double mutant cpc-1 ttg1-23,generated very few root hairs and exhibited increased ectopic expression of GL2::GUS as compared to cpc-1.The ttg1-23 single mutant did not exhibit an obvious defect in root epidermis development and the GL2::GUS expressing pattern is also similar to the wild type.Consistent with a mutation in the TTG1 gene,the ttg1-23 line also exhibited reduced seed coat pigmentation,altered trichome formation,and diminished seedling anthocyanin production.ttg1-23 mutation was a single G to A substitution at position 8,372,144 on chromosome 5 within the TTG1 gene,which changes the serine encoded by the 197 th codon to phenylalanine(S197F)in the predicted TTG1 protein.2.ttg1-24,derived from the same EMS-based screening as ttg1-23,was indentifed that resembled the ttg1-23 line and and bore a single C to T substitution in codon 339,which changes a leucine to a phenylalanine(L339F)near the C-terminus of the predicted 341 amino acid TTG1 protein.3.The mutated TTG1 proteins from ttg1-23 and ttg1-24 exhibited a diminished ability to interact with GL3 protein,which reduced the effectiveness of the MYB-b HLH-WD40 complex.The TTG1::ttg1-23-EYFP and TTG1::ttg1-24-EYFP proteins exhibited the same EYFP accumulation pattern as TTG1-EYFP,indicating that the ttg1-23 and ttg1-24 mutations had little effect on the cellular accumulation of the TTG1 protein.The double mutants,try ttg1-23,etc1 ttg1-23,try ttg1-24 and etc1 ttg1-24,did not generate obviously increased non-hair cell fate compared with each of the single mutant,indicating that ttg1-23 and ttg1-24 were not able to enhance the phenotype of try or etc1.4.The weak mutations(ttg1-9,ttg1-23 and ttg1-24)generated similar effect on enhancing the cpc-1 phenotype.Whereas,the strong mutations(ttg1-1 and ttg1-13),exhibiting a hairy phenotype,did not enhance cpc-1 but instead,they suppressed the cpc-1 mutant phenotype.The ttg1 mutations differentially affected target gene expression.The GL2 transcript level was dramatically reduced in the strong ttg1-1 and ttg1-13 mutants,whereas the weak ttg1-9,ttg1-23 and ttg1-24 showed almost normal GL2 levels.The weak ttg1 mutants(ttg1-9,ttg1-23,and ttg1-24),possessed a higher level of CPC RNA than the strong ttg1 mutants(ttg1-1,ttg1-13).5.In this study,two new allelic mutants of TTG1 gene,ttg1-23 and ttg1-24,were identified,which exhibited significantly different phenotype from the known ttg1 mutants,providing a new understanding of the function of TTG1 gene.ttg1-23 and ttg1-24 differentially regulated the expression of downstream genes to achieve their functional differences from known mutants.At the same time,the ttg1-23 and ttg1-24 mutations reduced the binding capacity between TTG1 and GL3 proteins,thus resulting in a change in the differentiation fate of epidermal cells.In addition,ttg1-23 and ttg1-24 single mutants had no obvious defects in root hair development,and the cpc-1 single mutant only partially inhibited root hair formation,while the cpc-1 ttg1-23 and cpc-1 ttg1-24 double mutants almost completely inhibited root hair development,indicating that the development of epidermal cells is controlled by multiple genes and is a complex regulatory process.The normally developed roots of crops,especially root hairs,directly affect the growth and development,disease resistance and yield.In this study,Arabidopsis was used as the model plant to discover the new alleles affecting the development of root epidermal cells,which further analyzed and improved the mechanism of root hair growth and development,providing an important theoretical basis and technical support for the idenditification of genes related to the control of root hair cell development in maize and other crops.