Oxylipin carbonyl compositions in the chloroplast compartments

Aldehydes and ketones derived from oxidized lipids (oxylipin carbonyls) accumulate in plants under various stress conditions, acting either as signaling molecules or as cytotoxic agents through their electrophilic modification of proteins and nucleic acids. Because chloroplasts generate reactive oxygen species and contain lipids rich in unsaturated fatty acids, they are likely major sites of oxylipin carbonyl formation in leaves. To identify chloroplast-derived carbonyl species and their suborganellar localization, we performed a comprehensive analysis of carbonyls in spinach leaves, whole chloroplasts, thylakoids, envelopes, and stroma. A total of 22 carbonyl species were detected, of which 12 were identified. The chloroplast contained 16 species, including formaldehyde, propionaldehyde, acetaldehyde, acetone, butyraldehyde, (E)-2-pentenal, (Z)-3-hexenal, (E)-2-hexenal, n-hexanal, and 4-hydroxy-(E)-2-hexenal. The thylakoid membrane contained all carbonyls found in the chloroplast except acetone, indicating that it is the primary site of carbonyl generation. Estimated concentrations of these carbonyls in the membrane reached approximately 2 mM. Importantly, our results demonstrate for the first time that formaldehyde is produced within chloroplasts and originates from membrane lipids. These findings provide new insight into the formation and compartmentalization of oxylipin carbonyls in plants.