Phosphorylation events controlling low oxygen-signaling in Arabidopsis thaliana
Final Report Abstract
Low oxygen stress triggers cellular signaling that culminates in activation of hypoxia responsive genes. Certain environmental conditions, such as heavy rain, waterlogging or flooding can cause low oxygen stress in plants due to the hampered diffusion of oxygen in water. The ERFVII transcription factor family is largely responsible for hypoxia response activation. Due to an N-terminal motif these transcription factors are targeted for proteasomal degradation by an oxygen dependent pathway which thus prevents the activation of hypoxia responsive genes in an oxygenated cellular environment. The ERFVII factors RAP2.12 is protected from this degradation pathway in oxygenated conditions through their association with ACYL-COA BINDING PROTEINS (ACBP). The first part this research project investigated how ACBP phosphorylation contributes to dissociation of RAP2.12 during low oxygen and focussed on identifying the kinases involved in this mechanism. In order to study ACBP1 phosphorylation status, a pull-down of ACBP1-GFP from transgenic plants treated with air and anoxic conditions was carried out followed by phosphoproteomic analysis of ACBP1. Two ACBP1 ankyrin domain peptides were found to be phosphorylated in anoxia and not in air samples. To investigate if ACBP1 phosphorylation could facilitate RAP2.12 dissociation, modified ACBP1 proteins were used in BiFC and affinity assays. Lastly, putative kinases responsible for ACBP1 phosphorylation during low oxygen were identified using kinase assays and a proximity labelling biotinylation technique. The second part of this research project investigated the ERFVII regulation by CIPK family members as well as the effect of this regulation on ERFVII activity. For that, members of the CIPK family that could interact with RAP2.2 and RAP2.12 were identified. Next, it was investigated which domain of RAP2.12 was responsible for the interaction with the CIPK. To research the effects of this interaction on RAP2.12 activity, the transcriptional activation of hypoxia responsive genes was observed in cipk mutants. Finally, to access the CIPK role during hypoxia, the levels of eleven hypoxia responsive genes were verified in cipk mutants under air and low oxygen conditions. To observe the physiological effects of an altered hypoxic gene expression in cipk mutants, low-oxygen survival assays were performed. The results of this project describes further sophisticated regulatory mechanisms to control the low-oxygen response pathways in plants. It provides convincing evidence that fine-tuning the initiation of energy saving adaptive responses to low-oxygen conditions is organized at many different molecular levels. Improving our understanding of these control mechanisms will not only provide us with better knowledge on the molecular regulation of stress responses in general, but will also help to design strategies to improve plant stress tolerance in a rapidly changing global environment.
