Construction And Screen Of An Arabidopsis EMS Mutant Library For The Study Of Auxin Reflux In The Root Tip

Arabidopsis is an excellent model for dicotyledonous plants. Plant roots are important organs for absorbing water and nutrients and for anchoring of the plant body to the ground. Auxin is a key plant hormone required for many aspects of root development, including initiation and emergence of lateral roots, patterning of the root apical meristem, gravitropism, and root elongation. A major determinant in auxin-mediated plant growth is the directed transport of auxin from sites of biosynthesis to target tissues. This transport mode is called polar auxin transport. Organ formation in Arabidopsis involves dynamic auxin gradients with maxima at the primordial tips. Auxin efflux, mediated by PIN family of auxin transporters, is crucial for the establishment of auxin gradients and maxima, and plays an important role in the regulation of auxin distribution in both aerial and underground organs. In the Arabidopsis root, disruption of auxin efflux is known to strongly affect the’auxin reflux’loop and thus interfere with the distal auxin maximum, altering the orientation and extent of cell division and root patterning.The mechanism controlling auxin reflux in the root remains unclear. To reveal genetic factors involved in this process, in this study we used a forward genetics approach to screen an EMS mutant library constructed in the background of WOX5::IAAH-DR5::GFP, which allows induction of auxin biosynthesis in the quiescent centre in the presence of the IAA precursor IAM, and changes in auxin accumulation and distribution can be monitored by using the DR5-GFP reporter. The main results of this study were described as follows:1. Screening condition was established for the needs of this study. M2plants were screened in the presence of exogenously applied IAM. Root hair density and DR5::GFP fluorescence intensity were taken as the early indicators of phenotypic changes, whereas primary root length, lateral root number and length, hypocotyl length and root gravitropism were taken as the later indicators.2.55candidate families were founded after screening and several rounds of rescreening. Segregation ratio of selfed progenies of candidate families was analyzed to determine whether they are homozygous. Homozygous candidate families were further divided into eight subgroups by their phenotypic similarity. 3. Phenotypes of candidate families were examined in the presence of auxins or polar auxin transport inhibitor NPA. By comparing the phenotypes caused by auxins, NPA and IAM, the possible causes of these mutant phenotypes are discussed.4. By analyzing gravity-induced root curvature, we found that all the candidate families had a WT-like response to gravity, except for these candidate families that showed gravitropism phenotype before reorientation.5. Genetic crosses were performed between candidate families in each of the subgroups. Based on the phenotypes of F1progenies, five pairs of allelic mutants were identified.