Functional studies of the Arabidopsis thaliana dormancy associated genes, DRM1 and DRM2

Abstract

Plants must carefully regulate their development in order to survive the prevailing conditions. One aspect of this of particular importance is dormancy release - meristematic tissues deciding when to grow and when not to, given varying conditions. In order to better understand the growth release mechanism of dormant tissue at the molecular and physiological levels molecular markers can be used. Members of the DRM1/ARP (DORMANCY ASSOCIATED GENE-1/AUXIN-REPRESSED PROTEIN) gene family are routinely used as markers for dormancy release. This plant-specific gene family has high sequence identity at the protein level throughout the plant kingdom, but its function in planta remains undetermined. The aim of this PhD project is to gain insight into the function of the dormancy associated genes DRM1 and DRM2 in Arabidopsis thaliana. A multi-faceted approach, including bioinformatic, molecular and biochemical studies, was adopted. The Arabidopsis DRM1/ARP gene family includes five members, the resulting proteins of which are predicted to be intrinsically disordered in nature. These family members are differentially regulated across development at the transcript level, with both constitutive and floral-specific, non-meristematic profiles evident. Both AtDRM1 and AtDRM2 produce splice variants which differ in their transcriptional response to various abiotic factors. Over-expression of AtDRM1 or AtDRM2 causes subtle developmental retardation. While null mutants do not exist for either gene, down-regulation amiRNA lines were analysed and exhibited subtle increases in bolt height compared with wildtype. Attempts to express His6-tagged AtDRM1 or AtDRM2 recombinant protein in E.coli yielded no expression, while expression attempts in a cell-free system produced an insoluble product. This protein is readily degraded in vitro, possibly as a result of its lack of intrinsic structure. Yeast-2-hybrid assays showed that neither AtDRM1 nor AtDRM2 bind with the branching pathway protein AtBRC1 and a lack of putative binding partners retrieved from a Yeast-2-hybrid library screen suggests that binding may be phosphorylation-dependent. Overall these findings lead to the hypothesis that AtDRM1 and AtDRM2 may be hub proteins acting in a general stress response pathway, specifically involved in the early modulation of protective growth prevention signals.