The role of a Vitis riparia dehydrin in protecting DNA from oxidative stress

Boddington, Kelly
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University of Guelph

Dehydrins are intrinsically-disordered proteins expressed during seed development and during abiotic stress. They have been shown to confer stress tolerance to plants, and many different methods of action have been proposed for these proteins. Here, I first show using both circular dichroism and NMR that the long-proposed nucleotide binding role of the Y-segment of dehydrins is incorrect. I then examine the expression, subcellular localization, and role of the YSK2-type Vitis riparia dehydrin, VrDHN1. Through phenol protein extraction studies, it was found that VrDHN1 was expressed during cold and dehydration stresses, as well as in response to the plant stress hormone ABA. Fluorescence-localization, confirmed by subcellular fractionation, revealed that VrDHN1 was found in the nucleus and in the cytoplasm of plant cells. The ability of VrDHN1 to bind to DNA in vitro was then investigated. VrDHN1 was shown to bind to DNA in a manner that was dependent on the sequence of VrDHN1, but not on the sequence of the DNA. This binding was not stimulated by metal ions, and was somewhat reduced by the pseudophosphorylation of the protein. Furthermore, the binding of VrDHN1 to DNA was shown to protect the DNA from oxidative damage caused by hydroxyl radicals, a function that was unhindered by the pseudophosphorylation of VrDHN1. In vivo experiments to confirm this protection of DNA were attempted, but were limited by difficulties in generating stable VrDHN1 transformants. I propose a model of DNA protection against oxidative damage, where VrDHN1 binds to DNA in a low-affinity and non-DNA sequence specific manner to reduce damage caused by an ROS attack.

DNA, dehydrin, reactive oxygen species, oxidative stress, Vitis riparia, electrophoretic mobility shift assay, plasmid isoform assay, NMR, subcellular localization, nucleus, VrDHN1, abiotic stress, cold stress, dehydration