The Nuclear Interactome of ATR7 Implicates a Chromatin-Based Repression Mechanism Controlling Oxidative Stress...

The redox state of the nucleus is emerging as a critical determinant of plant cell fate: reactive oxygen species (ROS) signals that originate in chloroplasts, peroxisomes, and the apoplast ultimately converge on nuclear proteins that determine whether a cell mounts a protective response or initiates programmed cell death (PCD). Loss-of-function mutations in ATR7, which encodes a nuclear protein specific to seed plants, confer tolerance to both paraquat- and aminotriazole-induced cell death, establishing ATR7 as a positive regulator of ROS-induced PCD. Yet how ATR7 acts at the molecular level remains unknown. In this paper, we define the ATR7 protein interactome using IP-MS of GFP-tagged ATR7 and integrate it with the atr7 loss-of-function transcriptome to distinguish it as candidate direct molecular partners from transcriptionally regulated targets. To investigate the nuclear protein association with ATR7, we performed GFP affinity purification followed by mass spectrometry (IP-MS) using Arabidopsis thaliana seedlings expressing GFP-ATR7, in comparison with seedlings expressing free GFP as the negative control. The IP-MS candidates were compared with the previously published ATR7 transcriptome data. ATR7 associates with chromatin-modifying proteins, components of the ubiquitin-proteasome system, and a broad set of stress-responsive proteins whose encoding genes are constitutively de-repressed when ATR7 is non-functional. Among the candidate proteins are those that have potential chromatin-regulatory functions, including AT1G01920 (a SET-domain protein) and HDA14, as well as components associated with ubiquitin-proteasome pathways and oxidative stress responses. Several interactors have no current functional annotation and represent candidates for novel roles in oxidative stress signalling. These findings provide the first mechanistic framework for ATR7 action and implicate nuclear chromatin-level repression as a key node in the regulation of ROS-induced PCD in plants.