ENOD93 Linked to Mitochondrial Function and Nitrogen Efficiency in Plants

By Dr. Chinta SidharthanReviewed by Lily Ramsey, LLMDec 6 2024

In nitrogen-fixing plants such as legumes, the protein EARLY NODULIN93 (ENOD93) plays an important role in nitrogen fixation during nodule development and is evolutionarily conserved. However, despite its importance, the molecular function of ENOD93 in cellular respiration remains unclear.

In a recent study published in Plant Cell, Australian and Canadian researchers explored the function of ENOD93 in Arabidopsis, focusing on its localization to the mitochondria and its impact on respiratory processes.

The findings revealed that ENOD93 influences mitochondrial energy production and root growth by interacting with cytochrome c oxidase (Complex IV). These results provided new insights into the relationship between mitochondrial function, nitrogen metabolism, and plant development, further emphasizing ENOD93’s broader significance beyond legumes.

​​​​​​​Study: EARLY NODULIN93 acts via cytochrome c oxidase to alter respiratory ATP production and root growth in plants. Image Credit: Leo Pakhomov/Shutterstock.com

Background

Mitochondria are fundamental to cellular energy generation, producing adenosine triphosphate (ATP) through oxidative phosphorylation. Cytochrome c oxidase (Complex IV) plays a pivotal role in this process by enabling electron transfer and maintaining membrane potential.

While significant progress has been made in understanding the protein components of mitochondrial complexes, the role of certain conserved proteins remains enigmatic.

Initially identified in leguminous plants during nodule development, ENOD93 is a conserved mitochondrial protein found in various plants and eukaryotes.

Early studies have reported its involvement in nitrogen fixation and nitrogen use efficiency, but its precise mitochondrial function was unclear.

Bioinformatics-based studies linked ENOD93 to the N-terminal domain of yeast respiratory supercomplex factor 2 (RCF2), a known Complex IV regulator. However, despite this link, direct evidence of the role of ENOD93 in mitochondrial processes and energy production in plants is lacking.

About the Study

The present study utilized Arabidopsis as a model to explore the role of ENOD93 in mitochondrial function. The researchers isolated an Arabidopsis mutant with a transfer deoxyribonucleic acid (T-DNA) insertion disrupting ENOD93 expression and complemented it with an ENOD93 complementary DNA to verify functional restoration.

Localization studies were conducted to confirm the presence of ENOD93 in mitochondria using mass spectrometry and targeted peptide analysis. The protein was found to be associated with cytochrome c oxidase (Complex IV) in mitochondria, but its abundance and activity in solubilized complexes remained unchanged across genotypes.

The researchers then conducted functional assays to assess mitochondrial respiration using oxygen electrode experiments. ATP synthesis and adenosine diphosphate (ADP) depletion rates were also analyzed in isolated mitochondria in the mutants with disrupted ENOD96 expression.

Additionally, safranin fluorescence assays were conducted further to confirm the elevated membrane potential in the mutant mitochondria and compare it to that of the wild-type and complemented lines.

Furthermore, whole-seedling respiration and root growth analyses were conducted to evaluate phenotypic outcomes. Metabolomic profiling was also performed to identify changes in the levels of organic and amino acids in mutant roots.

To examine the evolutionary conservation of ENOD93, the researchers also employed bioinformatics-based analyses using sequence alignments and hidden Markov models.

ENOD93 homologs were identified across diverse plant and microbial lineages, suggesting an ancient role in mitochondrial function. These integrated methods provided comprehensive insights into the role of ENOD93 in mitochondrial energy dynamics.

Major Findings

The study found that ENOD93 is critical for optimal mitochondrial function and energy production. Loss of ENOD93 activity in Arabidopsis resulted in a gradual decline in ADP-stimulated respiration and ATP synthesis rates, although it did not affect the abundance or basic activity of Complex IV in solubilized mitochondria. Instead, the defect was linked to an elevated mitochondrial membrane potential that restricted oxidative phosphorylation.

Furthermore, the whole-seedling respiration analyses showed higher respiration rates in the mutants with disrupted ENOD93 expression.

At the same time, root growth was more sensitive to ATP synthesis disruptions, highlighting the role of the protein in maintaining energy balance.

Additionally, metabolomic profiling revealed significantly higher levels of metabolites such as pyruvate, alanine, and fumarate in the roots of the mutants, reflecting altered metabolic fluxes. However, the complementation experiments restored mitochondrial and metabolic functions, confirming the role of ENOD93.

Moreover, ENOD93 was also found to be associated with Complex IV, suggesting its involvement in the electron transport chain. Elevated ADP levels and reduced ATP/ADP ratios in the roots of the mutants further indicated impaired energy regulation.

However, recovery of respiratory capacity upon dissipation of the membrane potential emphasized a Complex IV-dependent mechanism underlying these defects. These observations suggested that ENOD93 modulates the balance between membrane potential and ATP production efficiency.

Lastly, the phylogenetic analyses revealed that ENOD93 homologs are widely distributed across plants and other eukaryotes, which provided evidence for its evolutionary conservation. The findings also linked the function of ENOD93 to the N-terminal domain of yeast RCF2, supporting its role in Complex IV regulation.

Conclusions

Overall, the study elucidated the pivotal role of ENOD93 in mitochondrial respiration and energy regulation. By associating with Complex IV, ENOD93 was found to modulate oxidative phosphorylation and balance membrane potential and ATP synthesis.

Furthermore, its absence was found to disrupt energy metabolism and root growth in Arabidopsis. These findings suggested that ENOD93 was an evolutionarily conserved regulator of mitochondrial function. The study also provided a foundation for further research on its role in plant energy dynamics and broader eukaryotic lineages.