Compartment-specific oxylipin-derived carbonyl profiles in spinach chloroplasts.

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conclusionThe 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 C