Functional Analysis Of DC1 Protein Involved In Root Elongation And Salt Stress Response In Arabidopsis Thaliana

Roots are indispensable organs for plants to absorb water and nutrients from the soil.The development status of roots directly determines the growth and development of plants.DC1 proteins are rich in cysteine and histidine amino acids.They are a type of zinc finger proteins which are widely distributed in plants and play multiple functions during plant growth and development.These proteins are also involved in a variety of abiotic stress responses such as salt,drought,light and oxidation and so on.In Arabidopsis,the DC1 family contains nearly 150 members,however,the functions and mechanisms of these proteins still remain undiscovered.To reveal the function of this type of protein in root development,we isolated and characterized a DC1 gene which was predominantly expressed in roots,designated as AtDC1-1.AtDC1-1 is predominantly expressed in the transition zone between root meristem and elongation zone,and may be involved in the regulation of cell division and differentiation in root development.The present thesis mainly studied the function and regulatory network of AtDC1-1 gene in Arabidopsis primary root development.The results are as follows:1.Activity analysis of AtDCl-1 promoterIn order to analyze the promoter activity of AtDC1-1,a 4034 bp promoter sequence of AtDC1-1 gene was cloned and the vector pG2NBL-AtDC1-1p::ERYFP was constructed and then transformed Arabidopsis.Several homozygous lines of transgenic plants expressing AtDC1-1p::ERYFP were obtained.It was found that the yellow fluorescence was mainly concentrated in the epidermal cells and cortical cells in the transition zone between the meristem zone and the elongation zone in 5 DPG roots by using the confocal microscope.Strong signals were also detected in mature zones and in the root caps.This result indicated that the promoter of AtDC1-1 exhibit high activities in Arabidopsis root,especially in the transition zones.2.Phenotype analysis of dcl-1 mutantThree T-DNA insertion homozygous mutants of AtDC1-1 gene(dcl-la,dc1-1b,dc1-1c)were bought for studying its role in root development.Phenotype analysis revealed that the three mutants showed shorter primary roots at different degrees,comparing with wild type plants.Among them,the roots of dc1-1b mutants are shortest,only about 2/3 of WT at 5 DPG.Through microscope,we found that in the mutants the length of the meristem zones and the elongation zones are both shorter than that in WT.The shorter meristem zones are mainly due to a decrease in the number of cells,while the shorter elongation zone are mainly caused by the shortening of cells.Above data indicated that the knockdown or knockout of the AtDC1-1 gene may cause a higher cell differentiation rate than that of cell division,resulting shorter primary roots.3.AtDC1-1 overexpression transgenic Arabidopsis phenotype analysisWe constructed the pK7CWG2-AtDCl-1 overexpression vector and transformed it into Arabidopsis,and obtained multiple AtDCl-1 overexpressing transgenic Arabidopsis lines.Compared with WT,AtDC1-1 overexpressing transgenic plants grew faster and the primary roots were significantly longer than that of WT.The length of the meristem zones increased significantly with much more cells in this zone.However,the length of the elongation zone was still shorter than that of WT slightly.It indicate that overexpression of AtDC1-1 gene can promote cell division.4.RNA-Seq analysis of differentially expressed genes in 5 DPG roots of Atdc1-1b mutants and WT plantsTo reveal the mechanism of AtDC1-1 participating in root development,we analyzed differentially expressed genes in 5 DPG roots of wild-type and dc1-1b mutant by RNA-Seq technique.The transcriptome sequencing results showed that 23 genes were up-regulated and 61 genes were down-regulated in the dc1-1b mutant.Among the genes whose expression is up-regulated,there are two genes related to cytokinin degradation and metabolism,CKX3 and UGT85A1,which have significantly higher transcripts than wild type.In addition,the expression levels of CRF4,CRF3 and ARR3,three negative regulators of cytokinin,were also significantly increased.We speculated that the knockout of AtDC1-1 may lead to the degradation of CK as well as the decrease of CK reponse in root tips,resulting in the phenotype of CK deficiency in root development,including imbalance of cell division and differentiation,and obstruction of root development.5.Isolation and characterized of the regulatory TFs upstream and the interacting proteins of AtDC1-1To analyze the gene regulatory network of AtDC1-1 involved in Arabidopsis root development,we screened the transcription factor Library of Arabidopsis by yeast one-hybrid and yeast two-hybrid systems.It was found that three transcription factors CBF4,CBF1,and AtHB31,can bind to the promoter of AtDC1-1.At the same time,four interaction proteins of AtDC1-1 were also identified.They are IAA27,SINAT4,ZML2 and MYB-L.Among them,ZML2(ZIM-like 2)belongs to GATA ? subfamily and has similar structure with zinc finger protein family.It has been reported that ZML2 is involved in root development regulation,suggesting that ZML2 may play a role in plant growth and development together with AtDC1-1.6.AtDCl-1 is involved in the response of salt stress in ArabidopsisTo investigate whether AtDC1-1 is involved in the abiotic stress response of Arabidopsis,we performed ABA,high salt and mannitol stress treatment on dc1-1b mutant and overexpressing transgenic plants.The results showed that the dc1-1b mutant was sensitive to high salt treatment,while the overexpressing transgenic plants showed high tolerance to salt stress.It indicated that AtDC1-1 was involved in the salt stress response of plants.Transcriptome data also showed that many known salt stress response genes such as AZF2,ZAT10 and other genes were expressed in dc1-1b mutant at a lower level.The results of qRT-PCR showed that under the salt treatment conditions,the AZF2 and ZAT10 genes in the overexpressing plants were significantly up-regulated,and their expression levels were obviously higher than that in WT.And the expression of these genes were also slightly up-regulated in the mutants,but significantly lower than that in WT plants.These results indicate that AtDC1-1 is involved in the Arabidopsis salt stress response,and overexpression of this gene can enhance salt tolerance in plants.