Activation of alternative oxidase ensures carbon supply for ethylene and carotenoid biosynthesis during tomato fruit ripening

Tomato (Solanum lycopersicum) is a climacteric fruit displaying a peak of respiration at the onset of ripening accompanied by increased synthesis of ethylene and carotenoid pigments. Chromoplast and mitochondrial respiration participate at different stages of fruit ripening, but their in vivo regulation and function remains unclear. We determined the in vivo activities of the mitochondrial alternative oxidase (AOX) and cytochrome oxidase pathways and quantified the levels of respiratory- and ripening-related gene transcripts, primary metabolites and carotenoids in ripening tomato fruits with or without a functional chromorespiration. Furthermore, we carried out physiological, molecular and metabolic analyses of CRISPR-Cas9 mutants defective in AOX1a, the main AOX isoform up-regulated during tomato fruit ripening. We confirmed that PTOX-dependent chromorespiration is only relevant at late stages of ripening and found that in vivo AOX activity significantly increased at the breaker stage, becoming the main contributor to climacteric respiration when ripening is unleashed. This activation did not correlate with gene expression but was likely due to increased levels of AOX activators such as pyruvate (a metabolic precursor of carotenoids), 2-oxoglutarate and succinate. A strong alteration of ripening-related metabolites was observed in aox1a mutant fruits, highlighting a key role of the AOX pathway at the onset of ripening. Our data suggest that increased supply of TCA cycle intermediates at climacteric stage allosterically enhance AOX activity, thus allowing the reoxidation of NAD(P)H to ensure carbon supply for triggering ethylene and carotenoid biosynthesis.