Compartment-specific oxylipin-derived carbonyl profiles in spinach chloroplasts

The first compartment-resolved map of chloroplast carbonyls was established, revealing distinct carbonyl compositions and patterns of accumulation in thylakoids, envelopes, and stroma. Oxylipin-derived carbonyls, diverse aldehydes and ketones generated from oxygenated lipids, function as key cytotoxic agents and signaling molecules in oxidative stress responses of plants. However, the intracellular and sub-organellar sites at which individual carbonyl species occur remain poorly defined. Here, we analyzed carbonyl compositions in spinach (Spinacia oleracea L.) leaves, isolated chloroplasts, and three chloroplast subcompartments: thylakoids, envelopes, and stroma. Across these samples, 26 carbonyl species were detected, of which 13 were identified. The carbonyl profile of chloroplasts differed markedly from that of whole leaves: phenylacetaldehyde and n-pentanal, both abundant in leaves, were absent or detected only at trace levels in chloroplasts, indicating that they are largely non-chloroplast-derived. Acetaldehyde was also much less abundant in chloroplasts than in leaves, although a minor chloroplast-associated pool was detected mainly in thylakoids and stroma. Within chloroplasts, the three subcompartments exhibited distinct carbonyl signatures. Formaldehyde was enriched in thylakoids and envelopes, suggesting a membrane-associated source consistent with formation from chloroplast lipid oxidation. By contrast, acetone was preferentially associated with envelope membranes, whereas several C₆ aldehydes were abundant in thylakoids and stroma. Apparent local concentration estimates suggested that several carbonyls can reach millimolar ranges in chloroplast membranes, although these values should be interpreted as compartmental accumulation capacities rather than exact in vivo concentrations. These results provide a compartment-resolved chemical map of oxylipin-derived carbonyls in chloroplasts and establish a spatial framework for investigating their formation mechanisms and potential metabolic and signaling functions in plant cells.