Abstract Spontaneous mutations are rare in mitochondria and the lack of mitochondrial transformation methods has hindered genetic analyses. We show that a custom-designed RNA-binding pentatricopeptide repeat (PPR) protein binds and specifically induces cleavage of ATP synthase subunit1 (atp1) mRNA in mitochondria, significantly decreasing the abundance of the Atp1 protein and the assembled F1Fo ATP synthase in Arabidopsis (Arabidopsis thaliana). The transformed plants are characterized by delayed vegetative growth and reduced fertility. Five-fold depletion of Atp1 level was accompanied by a decrease in abundance of other ATP synthase subunits and lowered ATP synthesis rate of isolated mitochondria, but no change to mitochondrial electron transport chain complexes, adenylates, or energy charge in planta. Transcripts for amino acid transport and a variety of stress response processes were differentially expressed in lines containing the PPR protein, indicating changes to achieve cellular homeostasis when ATP synthase was highly depleted. Leaves of ATP synthase-depleted lines showed higher respiratory rates and elevated steady-state levels of numerous amino acids, most notably of the serine family. The results show the value of using custom-designed PPR proteins to influence the expression of specific mitochondrial transcripts to carry out reverse genetic studies on mitochondrial gene functions and the consequences of ATP synthase depletion on cellular functions in Arabidopsis.