Abstract
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 (H2O2), 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 H2O2 under untreated conditions.
Highlights
Mitochondria and plastids are composed of ;1500 and ;3000 proteins, respectively, with >95% of these proteins encoded by nuclear-located genes (Woodson and Chory, 2008)
Two-week-old Col:LUC seedlings, when treated with 50 μM antimycin A (AA), showed high expression of LUC driven by the ALTERNATIVE OXIDASE1a (AOX1a) promoter (Figures 1A to 1C)
Our results support a role for RAO2/ANAC017 in directly binding the promoter region of AOX1a to regulate transcription and shed light on the mechanism of signal transduction from signals perceived in the cytosol that release ANAC017 from its bound state within the endoplasmic reticulum (ER)/F-actin to the nucleus to affect a response to stress
Summary
Mitochondria 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 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). Plastid retrograde signals that have been identified more recently include 39-phosphoadenosine 59-phosphate (Estavillo et al, 2011), b-cyclocitral that is produced in plastids during high-light stress (Ramel et al, 2012), methylerythritol cyclodiphosphate, a precursor of isoprenoids (Xiao et al, 2012), and pathogen-associated molecular pathogen signals that are relayed via the chloroplasts through a Ca2+-mediated signal transduction pathway (Nomura et al, 2012)
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