We report the characterization of the Arabidopsis () 3-hydroxyacyl-acyl carrier protein dehydratase (mtHD) component of the mitochondrial fatty acid synthase (mtFAS) system， encoded by AT5G60335. The mitochondrial localization and catalytic capability of mtHD were demonstrated with a green fluorescent protein transgenesis experiment and by in vivo complementation and in vitro enzymatic assays. RNA interference (RNAi) knockdown lines with reduced expression exhibit traits typically associated with mtFAS mutants， namely a miniaturized morphological appearance， reduced lipoylation of lipoylated proteins， and altered metabolomes consistent with the reduced catalytic activity of lipoylated enzymes. These alterations are reversed when mutant plants are grown in a 1% CO atmosphere， indicating the link between mtFAS and photorespiratory deficiency due to the reduced lipoylation of glycine decarboxylase. In vivo biochemical feeding experiments illustrate that sucrose and glycolate are the metabolic modulators that mediate the alterations in morphology and lipid accumulation. In addition， both and mutants exhibit reduced accumulation of 3-hydroxytetradecanoic acid (i.e. a hallmark of lipid A-like molecules) and abnormal chloroplastic starch granules; these changes are not reversible by the 1% CO atmosphere， demonstrating two novel mtFAS functions that are independent of photorespiration. Finally， RNA sequencing analysis revealed that and mutants are nearly equivalent to each other in altering the transcriptome， and these analyses further identified genes whose expression is affected by a functional mtFAS system but independent of photorespiratory deficiency. These data demonstrate the nonredundant nature of the mtFAS system， which contributes unique lipid components needed to support plant cell structure and metabolism.