Isolation and Characterization of SCARECROW Suppressor Mutants in Arabidopsis thaliana
Type of DegreeThesis
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SCARECROW (SCR) is a transcriptional regulator that plays key roles in several developmental processes in a model plant Arabidopsis thaliana. SCR orthologs have been identified in agriculturally important plants such as rice and corn. It has also been demonstrated that at least some of the SCR functions have been conserved throughout plant kingdom. In Arabidopsis, among its many functions SCR is essential for the maintenance of root meristem, the development of root and shoot endodermis and for normal shoot gravitropism. As a result, scr mutants exhibit many phenotypic defects relating to these functions such as short roots, short hypocotyls, small leaves, shoot agravitropism as well as cell layer deletions in both roots and shoots. We have initiated a genetic screen to identify mutants that suppress scr mutant phenotype(s). In this scr suppressor screen we aim to identify and characterize other components of SCR-regulated pathways. Seeds homozygous for scr1 were mutagenized with EMS, germinated and grown to maturity. Seeds were collected in pools from approximately100 plants in each pool. All the seeds from the first 29 pools, representing approximately 1800 independent lines were screened for suppressor phenotypes. Over 200 potential suppressors were isolated. The majority of the putative suppressors had either longer roots or showed some degree of hypocotyl gravitropism. The potential suppressors were grown to maturity and seeds were collected from all the surviving and fertile plants. We collected seeds from 130 of the primary isolates. All of them were retested for suppressor phenotype and those that still had longer roots or gravitropic hypocotyls were tested for the presence of scr1 allele. Six of the primary isolates have been confirmed as scr suppressors. Five of these mutants display hypocotyl gravitropism and only one has a significantly increased root length. Five of the isolated suppressors represent at least two genes involved in hypocotyls gravitropism. Only one of the identified suppressors functions in root meristem maintenance. This suppressor, 24R1, was selected for mapping analysis. 24R1 plants were crossed to scr3, which is in a different genetic background. One quarter of the progeny from the cross has long roots indicating that the suppressor is not allele specific. DNAs isolated from individual plants with long root phenotype were used for mapping using molecular markers. Two to three Cleaved Amplified Polymorphic Sequences (CAPS) markers were used for each of the five Arabidopsis chromosomes. The only linkage was shown for chromosome 4. The mutation responsible for the suppressor phenotype in 24R1 maps to long arm of chromosome 4 about 21cM away from the closest CAPS marker available. This chromosomal region contains 22 genes that may correspond to 24R1 suppressor.