Genetic Analysis of DAWDLE Function in Arabidopsis
Narayanan, Lakshmi A.
CommitteeGordon, Donna M.
DAWDLE (DDL) is one of the eighteen genes in Arabidopsis thaliana that encodes a protein with a Fork-Head Associated domain, a phospho-threonine binding domain providing a role in DNA repair and cell cycle regulation. DDL also contains an arginine-rich N terminal domain with putative Nuclear Localization Signals and a region for RNA binding. Two ddl knockout alleles in the WS-2 ecotype exhibit a strong pleiotropic phenotype with developmental defects such as short root and hypocotyl, reduced fertility, and distorted organs. This developmentally delayed phenotype is due to reduced accumulation of microRNAs and the phenotype is similar to those displayed by mutants involved in microRNA biogenesis pathway, suggesting a function for DDL in the microRNA biogenesis. One of the goals of my research was to characterize DDL protein through a structure-function study. Twelve point mutants were isolated in a mutagenesis screen and a comparative phenotypic and molecular characterization of each mutant with wildtype (WT) plants was performed. This revealed a few functionally significant amino acid residues of DDL. Traits for comparison included hypocotyl and root length, plant height, fertility, and microRNA accumulation. While all the mutants displayed reduced fertility, some of them had significantly varying stem height, hypocotyl and root length, and microRNA accumulation compared to the WT. Another objective of my research was to identify components involved in the DDL pathway, which in-turn would contribute to discovering additional components in microRNA biogenesis pathway. One such component, ddl suppressor1 (dds1), was identified through a second site mutagenesis screen of ddl-2. Phenotypic and molecular characterization revealed that dds1 is a strong suppressor of ddl that was mapped on chromosome 3 of Arabidopsis. Another component identified was MODIFIER OF DDL (MDL), a natural variation between Col and WS-2 ecotype of Arabidopsis. The variation has been mapped to an interval consisting of 37 genes on chromosome 5. MDLCol is the dominant allele and imparts a weak phenotype to ddl knockouts, whereas the recessive MDLWS-2 does not modify a strong ddl knockout phenotype. ddl MDLCol does not display abnormality in microRNA accumulation unlike ddl MDLWS-2 indicating that MDL has a function related to microRNA biogenesis.