LIPOXYGENASE-2 OXYGENATES STORAGE LIPIDS IN EMBRYOS OF GERMINATING BARLEY

Besides the pre-existing lipoxygenase (LOX-1) present in quiescent gra ins, a new lipoxygenase (LOX-2) is induced in embryos of germinating b arley [Holtman. W. L., Van Duijn, G., Sedee, N. J. A. & Douman, A. C. (1996) Plant Physiol. 111, 569-576]. The fact that LOX-1 and LOX-2 for m different products after incubation with linoleic acid, the (9S)- an d (13S)-hydroperoxides, respectively [Van Aarle, P. G. M., De Barse, M . M. J., Veldink, G. A. & Vliegenthart, J. F. G. (1991) FEBS Lett. 280 , 159-162; Doderer. A., Kokkelink, I., Van der Veen, S., Valk, B. E. S chram, A. W. & Douma, A. C. (1992) Biochim. Biophys. Acta 1120, 97-104 ], and differ in temporal expression, suggests different physiological functions for the two isoenzymes at the onset of germination. We aime d to obtain more information about these functions by studying the sub strate and product specificities of both isoenzymes. Analyses of the p roducts formed from linoleic acid confirmed that LOX-1 oxygenated at C 9, and LOX-2 at C13. when testing more complex substrates, it was foun d that both LOX-1 and LOX-2 were capable of metabolizing esterified fa tty acids. K-m values from both isoenzymes for free fatty acids wee mu ch lower than for esterified fatty acids (7-35-fold for LOX-1 versus 2 -8-fold for LOX-2). Interestingly, LOX-1 showed significantly higher K -m values for esterified fatty acids than did LOX-2. This was reflecte d by analyses of the products formed from di- and tri-linoleoylglycero l; LOX-2 formed higher amounts of oxygenated polyunsaturated fatty aci ds within the esterified lipids than did LOX-1, with a corresponding l arger extent of oxygenation.In order to identify potential endogenous substrates, we analyzed free and esterified lipids in total lipid extr acts from barley after different periods of germination for LOX-derive d products. The results indicated that esterified fatty acids were pre ferentially metabolized by. LOX-2 activity, Analysis of the positional specificity within the lipids after alkaline hydrolysis revealed that only (13S)-hydroxy derivatives were formed. indicating the in vitro a ction of LOX-2. These data show that LOX-2 is capable of oxygenating s torage lipids and suggest that during the onset of germination LOX-2 m ay be involved in oxygenation of esterified polyunsaturated fatty acid s in barley seeds. We suggest that the oxygenation of these lipids pre cedes the onset of their catabolism and that the degradation product, (9Z,11E,13S)-13-hydroxy-octadecadienoic acid, serves as an endogenous substrate for beta-oxidation ind therefore as a carbon source for the growing bailey embryo.