Complex I Dysfunction Redirects Cellular and Mitochondrial Metabolism in Arabidopsis

Marie Garmier,Corinne Vallet,Ian D Small,A Harvey Millar,Etienne Delannoy,Adam J Carroll,David A Day

doi:10.1104/pp.108.125880

Abstract

Mitochondrial complex I is a major avenue for reduced NAD oxidation linked to oxidative phosphorylation in plants. However, the plant enzyme has structural and functional features that set it apart from its counterparts in other organisms, raising questions about the physiological significance of this complex in plants. We have developed an experimental model in which rotenone, a classic complex I inhibitor, has been applied to Arabidopsis (Arabidopsis thaliana) cell suspension cultures in order to dissect early metabolic adjustments involved in cell acclimation to mitochondrial dysfunction. Rotenone induced a transitory decrease in cellular respiration (0-4 h after treatment). Cell respiration then progressively recovered and reached a steady state at 10 to 12 h after treatment. Complex I inhibition by rotenone did not induce obvious oxidative stress or cell death but affected longer term cell growth. Integrated analyses of gene expression, the mitochondrial proteome, and changes in primary metabolism indicated that rotenone treatment caused changes in mitochondrial function via alterations in specific components. A physical disengagement of glycolytic activities associated with the mitochondrial outer membrane was observed, and the tricarboxylic acid cycle was altered. Amino acid and organic acid pools were also modified by rotenone treatment, with a marked early decrease of 2-oxoglutarate, aspartate, and glutamine pools. These data demonstrate that, in Arabidopsis cells, complex I inhibition by rotenone induces significant remodeling of metabolic pathways involving the mitochondria and other compartments and point to early metabolic changes in response to mitochondrial dysfunction.

Highlights

Mitochondrial complex I is a major avenue for reduced NAD oxidation linked to oxidative phosphorylation in plants
A heterotrophic cell suspension culture of Arabidopsis was grown in the dark until its midlog growth phase and treated with either 40 mM rotenone dissolved in methanol (0.25%, v/v) or methanol as a control
To assess the longevity of the impact of rotenone on respiration, the respiratory rate of cells treated for 48 h with rotenone was measured with or without a further rotenone addition (Supplemental Fig. S1, A and B), revealing that the pretreated cells were resistant to further rotenone treatments

Summary

Introduction

Mitochondrial complex I is a major avenue for reduced NAD oxidation linked to oxidative phosphorylation in plants. CMSII plants lack rotenone-sensitive complex I activity and structure and show a stable inherited phenotype involving delayed germination and development, light-dependent cytoplasmic male sterility, decreased photosynthetic efficiency, modified light acclimation responses, and altered organic acid and amino acid pools and antioxidant defenses (Sabar et al, 2000; Dutilleul et al, 2003a, 2003b, 2005) This mutant shows enhanced expression of the cyanideinsensitive alternative oxidase (AOX) and enhanced resistance to ozone damage and tobacco mosaic virus infection, suggesting a complicated link between mitochondrial metabolism, cellular redox regulation, and plant stress tolerance mechanisms (Dutilleul et al, 2003b; Vidal et al, 2007)

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