Search

Saturated and unsaturated N-acylethanolamines (NAEs) occur in desiccated seeds primarily as 16C and 18C species with N-palmitoylethanolamine and N-linoleoylethanolamine (NAE 18:2) being most abundant. Here, we examined the metabolic fate of NAEs in vitro and in vivo in imbibed cotton (Gossypium hirsutum) seeds. When synthetic [1-(14)C]N-palmitoylethanolamine was used as a substrate, free fatty acids (FFA) were produced by extracts of imbibed cottonseeds. When synthetic [1-(14)C]NAE 18:2 was used as a substrate, FFA and an additional lipid product(s) were formed. On the basis of polarity, we presumed that the unidentified lipid was a product of the lipoxygenase (LOX) pathway and that inclusion of the characteristic LOX inhibitors nordihydroguaiaretic acid and eicosatetraynoic acid reduced its formation in vitro and in vivo. The conversion of NAE 18:2 in imbibed cottonseed extracts to 12-oxo-13-hydroxy-N-(9Z)-octadecanoylethanolamine was confirmed by gas chromatography-mass spectrometry, indicating the presence of 13-LOX and 13-allene oxide synthase, which metabolized NAE 18:2. Cell fractionation studies showed that the NAE amidohydrolase, responsible for FFA production, was associated mostly with microsomes, whereas LOX, responsible for NAE 18:2-oxylipin production, was distributed in cytosol-enriched fractions and microsomes. The highest activity toward NAE by amidohydrolase was observed 4 to 8 h after imbibition and by LOX 8 h after imbibition. Our results collectively indicate that two pathways exist for NAE metabolism during seed imbibition: one to hydrolyze NAEs in a manner similar to the inactivation of endocannabinoid mediators in animal systems and the other to form novel NAE-derived oxylipins. The rapid depletion of NAEs by these pathways continues to point to a role for NAE metabolites in seed germination.

Lipoxygenases 1 and 2 were localized in etiolated germinating soybean seeds (Glycine max [L.]. Merr. var. Williams) by an indirect immunofluorescence staining technique. Sections of paraffin-embedded seedlings were stained with affinity-purified antibodies directed against lipoxygenase 1 or 2. The specificity of the immunofluorescence technique was examined by use of nonimmune serum or immunoglobulin G preparations after total adsorption with the appropriate lipoxygenase coupled to Sepharose 4B.After immunofluorescence staining with antilipoxygenase 1 or 2 IgG storage tissues of cotyledons fluoresce strongly the first days of germination. After 3 days, the abaxial hypodermis, the epidermis, and the vascular bundle sheaths show fluorescence, especially after incubation with antilipoxygenase 2 IgG. Fluorescence in cortex and pith of the hypocotyl migrates to the vascular cylinder during germination. In primary leaves, all tissues show fluorescence after 1 day of germination. In storage tissues of cotyledons, cytoplasm around the protein bodies fluoresces, whereas in other tissues protein bodies or other large cell organelles fluoresce.It is reasonable to suggest that lipoxygenase exerts its function in cells at the time that rigorous changes in metabolism take place, namely at the start of mobilization of reserves in storage tissues and start of biosynthesis of chloroplastids in several tissues.

Soybean lipoxygenases-1 and -2 were localized intracellularly in seeds at various stages of germination by indirect labeling of cryosections with protein A-colloidal gold complexes. Two sizes of gold particles (Au(5) and Au(16)) were used in single- and double-labeling experiments. In primary leaves, lipoxygenases are demonstrated to occur in vacuolating parenchyma cells but not in massive, nondifferentiated cells. In cotyledons, both isoenzymes are localized in the cytoplasm of storage parenchyma cells and in an aberrant type of protein bodies, occurring in hypodermis and vascular bundle sheath cells. No association has been found with either protein bodies in storage parenchyma cells or lipid bodies, mitochondria, and other organelles in any type of cell. The possible significance of lipoxygenase in the metabolism of storage lipids and its possible function as a regulatory enzyme are discussed on the basis of the random distribution throughout the cytoplasm of storage parenchyma cells and the course of biochemical processes during seed germination.