A functional genomic and genetic analysis of subclass III Actin Depolymerizing Factor (ADF) proteins in Arabidopsis thaliana
King, Lori Marie
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The Actin Depolymerizing Factor (ADF)/Cofilin family of proteins are essential actin-binding proteins found in all eukaryotes. ADF proteins modulate actin filament dynamics by severing monomers from the pointed end of the filaments. The Arabidopsis thaliana genome encodes 11 ADF proteins, which group into 4 subclasses. Mutants of the two ADF proteins in Subclass III, ADF5 and ADF9, have opposite flowering-time phenotypes: adf5-1 flowers with more rosette leaves than wild-type plants grown under the same conditions (16h light/8h night photoperiod), while adf9-1 flowers with fewer rosette leaves than wild-type. Also, qRT-PCR assays show that the transcript levels of the central repressor of flowering in Arabidopsis, Flowering Locus C (FLC), are up (fold-change: 4±0.2) in adf5-1 and down (fold change: 5±0.5) in adf9-1. My objective in this study was to determine how ADF5 and ADF9 proteins can change gene expression. Using a microarray analysis, I determined that ADF5 and ADF9 change the expression of approximately 7% and 12% respectively (false discovery rate of 0.1) of the genes expressed in the tissues (shoot) and at the time (2-4 leaves visible) I tested. Using genetic analyses focused on suppression studies, I show that the change in FLC expression is actin- dependent. Treatment with the actin filament disruptor Cytochalasin D is sufficient to increase FLC expression, suggesting that actin-cytoskeletal dynamics play a regulatory role in FLC expression. I overexpressed a wild-type polymerizable form of ACTIN2 in adf9-1 and showed that early flowering and the down-regulation of FLC is suppressed in this mutant by increasing the actin monomeric pool.