OsPUB75, an Armadillo/U-box protein interacts with GSK3 kinase and functions as negative regulator of abiotic stress responses

Abstract

It has become increasingly evident that the protein degradation mediated by E3 ligases is a crucial cellular process that regulates both biotic and abiotic stresses in plants. Previously, we have reported the microarray expression analysis of ARM/U-box proteins during abiotic stress conditions. In this study, we have performed a detailed functional analysis of OsPUB75/ARM, which is transcriptionally repressed during stress conditions. OsPUB75/ARM is a cytosolic RING-type E3 ubiquitin (Ub) ligase that negatively regulates drought and salinity induced osmotic stress response in Arabidopsis. The phenotypic analysis of OsPUB75/ARM-OE showed inhibition of growth due to enhanced ROS production during salinity and mannitol induced oxidative stress conditions. Yeast two-hybrid screening using OsPUB75/ARM as bait identified OsSK41 (Shaggy-like kinase 41) as its target, which belongs to the Glycogen Synthase kinase 3 (GSK3) family. A specific interaction between OsPUB75/ARM and OsSK41 and auto-ubiquitination activity of OsPUB75/ARM were demonstrated in planta and in vitro. The subcellular localization and protein interaction by BiFC assay in Nicotiana benthamiana showed that OsPUB75/ARM and OsSK41 co-localize to the cytosolic spherical body, which lies in close proximity to the plasma membrane. The sensitivity of OsPUB75/ARM-OE lines on stress media indicates that this protein might be involved in the negative regulation of antioxidant system, which sets the redox-status of the cell upon activation by OsSK41. Collectively, our results suggest that OsPUB75/ARM is an important negative regulator of abiotic stress responses in Arabidopsis.