Oxylipin carbonyl compositions in the chloroplast compartments
Aldehydes and ketones derived from oxidized lipids (oxylipin carbonyls) accumulate in plants under stress conditions, acting as signaling molecules or cytotoxic agents through electrophilic modification of biomolecules. Because chloroplasts generate reactive oxygen species and contain lipids rich in unsaturated fatty acids, they are likely major sites of oxylipin carbonyl formation. To identify chloroplast-derived carbonyl species and their sub-organellar distribution, we analyzed carbonyls in spinach leaves, whole chloroplasts, thylakoids, envelopes and stroma. A total of 26 carbonyl species were detected, of which 13 were identified. Chloroplasts contained 14 species, including formaldehyde, propionaldehyde, acetaldehyde, acetone, butyraldehyde, (E)-2-pentenal, (Z)-3-hexenal, (E)-2-hexenal, n-hexanal and 4-hydroxy-(E)-2-hexenal. Phenylacetaldehyde and n-pentanal, which were abundant in leaves, were not associated with chloroplasts. Thylakoid membranes contained all chloroplast carbonyls except acetone, indicating that they are a major site of carbonyl formation. Formaldehyde was highly enriched in thylakoids and envelopes, providing evidence that it can be generated within chloroplasts, most likely from membrane lipids. Estimated concentrations of several carbonyls in membranes reached over 1 millimolar. This high-resolution mapping of oxylipin carbonyls within chloroplast compartments, achieved here for the first time, provides a spatial framework for understanding their formation and functional roles in plant stress responses.