Molecular Mechanism Of DOFL1 Regulated Lateral Root Growth And Development In Arabidopsis

For plants,root is the important organ which can maintain the development of plants.It plays a curial role in anchoring plants,absorbing water and nutrients.Lateral roots,which account for a large proportion of root system,can enhance the attachment ability of the root system and the ability to absorb water and nutrients,at the same time it can respond to various environmental signals and affect the growth and development of the plant.Therefore,the investigation of the molecular mechanism and signaling regulation machinery about lateral root development has both theoretical and practical significance.In Arabidopsis,the development of lateral root is regulated by auxin,ethylene and cytokinin.The IAA14-ARF7/ARF19-LBDs auxin signaling circuit is the most profoundly investigated signaling pathway for the regulation of lateral root growth.However,the molecular mechanism underlying how lateral root is regulated is not understood very well.This study is devoted to identifying new regulators in lateral root development,and address how these regulators control lateral root development.DOF proteins are members of a major family of plant transcription factors,and composed of 200-400 amino acids with a N-terminal highly conserved DNA-binding domain and a C-terminal transcriptional activation domain.In recent years,DOF domain have been isolated from maize and other plant species,including Arabidopsis,tobacco,pumpkin,potato,barley,wheat and rice.Recent studies have shown that the DOF protein plays an important role in light signaling responses,plant hormones,defense responses,seed germination,the growth and development of the plant.In this study,the model plant Arabidopsis thaliana was used as the research object to identify new factors which can interact with LBD18 and regulate lateral root development.DOFL1,which is a DOF transcription factor,was found to interact with LBD18.The molecular mechanism by which DOFL1 controls lateral root development was investigated using molecular biology,cell biology,genetics,physiology and other techniques,and we have come to the following conclusions.1.In dofll mutant plants,a reduced lateral root primordia,an increased lateral root emergence and reduced total lateral roots were detected.In 35S::DOFLl transgenic plants,an increased lateral root primordia,a reduced lateral root emergence and increased total lateral roots were observed,indicating that DOFL1 can promote the lateral root priming.2.To assess the expression pattern and subcellular localization of DOFL1,we generated pDOFL1::GUS and 35S::GFP-DOFL1 lines.GUS staining analysis of several pDOFL1::GUS transgenic lines revealed that DOFL1 was expressed in both roots and leaves in Arabidopsis.Strong DOFL1::GUS signals were detected in root pericycles,various developmental stages of lateral roots and leaf vascular bundle cells.Through Agrobacterial mediated plant transformation in tobacco,DOFL1::GFP fluorescence signals were mainly detected in the nucleus of tobacco leaf cells.3.It is well known that ARF7 acts upstream of LBD18,we further tested if ARF7 could regulate the transcription of DOFL1,which encodes a protein interacting with LBD18.Our Q-PCR analysis showed that ARF7 could promote the expression of DOFL1.However,the yeast one hybrid analysis and transient expression assays in protoplast cells indicate that ARF7 doesn't interact with DOFL1 promoter directly,implying the indirect regulation of DOFL1 expression by ARf7.4.GATA23 was reported to be expressed in lateral root primordia and founder cells.The expression of GATA23 is dependent on the regulation of IAA28-ARF7-ARF19 signaling pathway.To address if DOFL1 can affect the transcriptional expression of GATA23,we tested the expression of GATA23 in 35S::DOFL1 transgenic plants,and found that GATA23 expression was increased compare to the WT control.Furthermore,the transient assays in protoplast and yeast one hybrid experiments demonstrate that the transcription factor DOFL1 directly regulates the transcription of GATA23.5.LBD18 was reported directly binding to the promoter of E2Fa and regulated its expression.Since DOFL1 was identified to interact with LBD18,it will be interesting to address if DOFL1 can affect the transcriptional expression of E2Fa.To this end,we tested the expression of E2Fa in 35S::DOFL1 transgenic plants through Q-PCR analysis,found that the expression of E2Fa is up-regulated compared to the WT control,suggesting that DOFL1 can promote the expression of E2Fa.The transient assays in protoplast and yeast one hybrid experiments further demonstrated that DOFL1 directly regulate the expression of E2Fa through binding to its promoters.This study indicates that LBD18 interacts with DOF transcription factor DOFL1,which can promote lateral root development.Auxin can induce the expression of DOFL1,which is mediated by ARF7 in an indirect manner.While DOFL1 can promote the expression of GATA23 and E2Fa directly.LBD18 regulates the expression of GATA23 and E2Fa through interaction with DOFL1.In summary,a DOF transcription factor DOFL1 was identified to interact with LBD18 and promote lateral root development through the regulation of GATA23 and E2Fa.