EARLY NODULIN93 acts via cytochrome c oxidase to alter respiratory ATP production and root growth in plants

Abstract

EARLY NODULIN 93 (ENOD93) has been genetically associated with biological nitrogen fixation in legumes and nitrogen use efficiency in cereals, but its precise function is unknown. We show that hidden Markov models define ENOD93 as a homolog of the N-terminal domain of RESPIRATORY SUPERCOMPLEX FACTOR 2 (RCF2). RCF2 regulates cytochrome oxidase (CIV), influencing the generation of a mitochondrial proton motive force in yeast (Saccharomyces cerevisiae). Knockout of ENOD93 in Arabidopsis (Arabidopsis thaliana) causes a short root phenotype and early flowering. ENOD93 is associated with a protein complex the size of CIV in mitochondria, but neither CIV abundance nor its activity changed in ruptured organelles of enod93. However, a progressive loss of ADP-dependent respiration rate was observed in intact enod93 mitochondria, which could be recovered in complemented lines. Mitochondrial membrane potential was higher in enod93 in a CIV-dependent manner, but ATP synthesis and ADP depletion rates progressively decreased. The respiration rate of whole enod93 seedlings was elevated, and root ADP content was nearly double that in wild type without a change in ATP content. We propose that ENOD93 and HYPOXIA-INDUCED GENE DOMAIN 2 (HIGD2) are the functional equivalent of yeast RCF2 but have remained undiscovered in many eukaryotic lineages because they are encoded by two distinct genes.