Plants require daily coordinated regulation of energy metabolism for optimal growth and survival and therefore need to integrate cellular responses with both mitochondrial and plastid retrograde signaling. Using a forward genetic screen to characterize regulators of alternative oxidase1a (rao) mutants, we identified RAO2/Arabidopsis NAC domain-containing protein17 (ANAC017) as a direct positive regulator of AOX1a. RAO2/ANAC017 is targeted to connections and junctions in the endoplasmic reticulum (ER) and F-actin via a C-terminal transmembrane (TM) domain. A consensus rhomboid protease cleavage site is present in ANAC017 just prior to the predicted TM domain. Furthermore, addition of the rhomboid protease inhibitor N-p-Tosyl-L-Phe chloromethyl abolishes the induction of AOX1a upon antimycin A treatment. Simultaneous fluorescent tagging of ANAC017 with N-terminal red fluorescent protein (RFP) and C-terminal green fluorescent protein (GFP) revealed that the N-terminal RFP domain migrated into the nucleus, while the C-terminal GFP tag remained in the ER. Genome-wide analysis of the transcriptional network regulated by RAO2/ANAC017 under stress treatment revealed that RAO2/ANAC017 function was necessary for >85% of the changes observed as a primary response to cytosolic hydrogen peroxide (H 2 O 2 ), but only ;33% of transcriptional changes observed in response to antimycin A treatment. Plants with mutated rao2/anac017 were more stress sensitive, whereas a gain-of-function mutation resulted in plants that had lower cellular levels of H 2 O 2 under untreated conditions. INTRODUCTIONMitochondria and plastids (chloroplasts) are composed of ;1500 and ;3000 proteins, respectively, with >95% of these proteins encoded by nuclear-located genes (Woodson and Chory, 2008). It has been shown that two-way communication pathways exist between the nucleus and mitochondria and chloroplasts, called anterograde and retrograde signaling pathways (Rhoads and Subbaiah, 2007;Woodson and Chory, 2008). Anterograde regulation refers to a top-down regulatory pathway, where signals have a direct impact on gene expression in the nucleus. Conversely, nuclear gene expression is also influenced by signals that originate from within the organelles, mitochondria, or chloroplasts and is referred to as retrograde regulation.Several components involved in plastid retrograde signaling have been identified, with at least five different pathways characterized: reactive oxygen species (ROS), redox signals, plastidial gene expression, pigment biosynthesis, and specific signaling metabolites (Pfannschmidt, 2010). The most intensively studied retrograde signaling pathway is in the genomes uncoupled (gun) mutants that uncouple the expression of nuclear-encoded chloroplastic proteins from the functional state of chloroplasts (Susek et al., 1993). A recently identified plastid-bound transcription factor, PTM (plant homeodomaintype transcription factor with transmembrane [TM] domains), was also identified as a regulator for plastid retrograde signaling and acts do...

Upon disturbance of their function by stress, mitochondria can signal to the nucleus to steer the expression of responsive genes. This mitochondria-to-nucleus communication is often referred to as mitochondrial retrograde regulation (MRR). Although reactive oxygen species and calcium are likely candidate signaling molecules for MRR, the protein signaling components in plants remain largely unknown. Through meta-analysis of transcriptome data, we detected a set of genes that are common and robust targets of MRR and used them as a bait to identify its transcriptional regulators. In the upstream regions of these mitochondrial dysfunction stimulon (MDS) genes, we found a cis-regulatory element, the mitochondrial dysfunction motif (MDM), which is necessary and sufficient for gene expression under various mitochondrial perturbation conditions. Yeast one-hybrid analysis and electrophoretic mobility shift assays revealed that the transmembrane domain–containing NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON transcription factors (ANAC013, ANAC016, ANAC017, ANAC053, and ANAC078) bound to the MDM  cis-regulatory element. We demonstrate that ANAC013 mediates MRR-induced expression of the MDS genes by direct interaction with the MDM  cis-regulatory element and triggers increased oxidative stress tolerance. In conclusion, we characterized ANAC013 as a regulator of MRR upon stress in Arabidopsis thaliana.