The sulfur dioxygenase ETHE1 oxidizes persulfides in the mitochondrial matrix and is involved in the degradation of L-cysteine and hydrogen sulfide. ETHE1 has an essential but as yet undefined function in early embryo development of Arabidopsis thaliana. In leaves, ETHE1 is strongly induced by extended darkness and participates in the use of amino acids as alternative respiratory substrates during carbohydrate starvation. Thus, we tested the effect of darkness on seed development in an ETHE1 deficient mutant in comparison to the wild type. Since ETHE1 knock-out is embryo lethal, the knock-down line ethe1-1 with about 1% residual sulfur dioxygenase activity was used for this study. We performed phenotypic analysis, metabolite profiling and comparative proteomics in order to investigate the general effect of extended darkness on seed metabolism and further define the specific function of the mitochondrial sulfur dioxygenase ETHE1 in seeds. Shading of the siliques had no morphological effect on embryogenesis in wild type plants. However, the developmental delay that was already visible in ethe1-1 seeds under control conditions was further enhanced in the darkness. Dark conditions strongly affected seed quality parameters of both wild type and mutant plants. The effect of ETHE1 knock-down on amino acid profiles was clearly different from that found in leaves indicating that in seeds persulfide oxidation interacts with alanine and glycine rather than branched-chain amino acid metabolism. Sulfur dioxygenase deficiency led to defects in endosperm development possibly due to alterations in the cellularization process. In addition, we provide evidence for a potential role of persulfide metabolism in abscisic acid (ABA) signal transduction in seeds. We conclude that the knock-down of ETHE1 causes metabolic re-arrangements in seeds that differ from those in leaves. Putative mechanisms that cause the aberrant endosperm and embryo development are discussed.