ECERIFERUM7 subunit of the exosome, SUPERKILLER complex and small RNA species regulate cuticular wax biosynthesis in Arabidopsis thaliana stems

The primary aerial surfaces of higher plants are covered by a continuous hydrophobic lipid layer called the cuticle, which is synthesized by the epidermal cells and provides protection against desiccation and environmental stresses. The cuticle is mainly composed of the cutin polyester matrix and cuticular waxes. Although the biosynthetic pathways of cuticular waxes are relatively well documented, how wax biosynthesis is regulated is not completely understood. The major goal of my thesis was to investigate the ECERIFERUM7 (CER7)-mediated mechanism of regulation of cuticular wax biosynthesis in stems of Arabidopsis thaliana. In particular, I was interested in investigating that how the Arabidopsis CER7 protein, a core component of the exosome complex that determines cellular RNA levels, was involved in this process. CER7 was proposed to degrade an mRNA encoding a repressor of wax biosynthetic gene CER3 to activate CER3 transcription required for stem wax biosynthesis. To identify the CER3 repressor and additional components of CER7 regulatory pathway, I carried out a cer7 suppressor screen and isolated mutants capable of restoring wild-type stem wax loads in the absence of CER7 activity. Characterization of these suppressor mutants and cloning of the affected genes resulted in a series of discoveries. First, cloning of RNA DEPENDENT RNA POLYMERASE 1 and SUPPRESSOR OF GENE SILENCING 3 from the suppressors demonstrated that small interfering RNAs (siRNAs) participate in CER7-mediated regulation of wax formation (Chapter 2). Second, forward genetics and reverse genetics, combined with small RNA sequencing confirmed that trans-acting siRNAs (tasiRNAs) are direct regulators of CER3 gene in CER7-controlled wax biosynthetic pathway (Chapter 3). Third, CER7 and tasiRNA-mediated regulation of CER3 during stem wax deposition requires the SUPERKILLER complex, which is known to be involved in cytoplasmic activities of the exosome in yeast and metazoan (Chapter 4).Science, Faculty ofBotany, Department ofGraduat