Lipoxygenase, Hydroperoxide Isomerase, and Hydroperoxide Cyclase in Young Cotton Seedlings

Abstract

Lipoxygenase was demonstrated in young cotton seedlings. It catalyzed the oxygenation of linoleic or linolenic acid, predominantly at carbon 13, and its molecular weight was estimated by gel filtration to be 100,000. Hydroperoxide isomerase was also present and converted hydroperoxy-linoleic or hydroperoxylinolenic acid to a-or y-ketols. The enzyme utilized the 13-hydroperoxy isomer in preference to the 9 isomer and its molecular weight was estimated at 250,000 by gel filtration. In addition, hydroperoxide cyclase, which catalyzes the conversion of 13-hydroperoxylinolenic acid to 12-oxo-phytodienoic acid, was present. Hydroperoxide isomerase and hy-droperoxide cyclase activities could not be separated by gel filtration and ion-exchange chromatography experiments, indicating the two enzyme activities may be associated with the same protein. The activities of all three enzymes were very low in the seed but increased immediately after germination, reached a maximum after 3 to 4 days, and then declined. The results suggest a role, as yet unknown, for these enzymes during early plant development. Lipoxygenase, hydroperoxide isomerase, and hydroperoxide cy-clase are enzymes which transform certain polyunsaturated fatty acids to oxygenated metabolites (Fig. 1). Lipoxygenase (EC 1.13.11.12) catalyzes the oxygenation of fatty acids containing a cis-l,cis-4-pentadiene structure to fatty acid hydroperoxides. The enzyme is present in a wide range of plant tissues (1) and, in the last few years, has been demonstrated in some animal tissues (2, 9, 10, 16, 19). Plant lipoxygenase is active with a number of polyunsaturated fatty acids in the 18-to 22-carbon range (13), but the natural substrate for the plant enzyme is probably linoleic or linolenic acid, the two most abundant polyunsaturated fatty acids in plant tissues. In vitro experiments have shown that these two acids are converted to either n-6 or n-10 hydroperoxides, depending upon the enzyme source, isoenzyme composition, pH, or 02 concentration (3). In animal tissues, lipoxygenase can catalyze the oxygenation of linoleic acid to the n-6 or n-10 hydroperoxides as in rat testes (19), or the oxygenation of arachidonic acid to the n-9 hydroperoxide as in human (10) or bovine (16) blood platelets. Lipoxygenase from rabbit leukocytes oxygenates the n-16 carbon of arachidonic acid (2). Hydroperoxide isomerase catalyzes the conversion of fatty acid hydroperoxides to a-or y-ketol compounds. The enzyme was first reported in flaxseed by Zimmerman (27) and was later demonstrated in several other plants (4, 6, 23, 26). Grossman et al. (8) have recently reported evidence for hydroperoxide isomerase in a 'This work conducted in cooperation with the North Dakota Agricultural Experiment Station, Paper No. 1061. microsomal preparation from rat testes. Hydroperoxide cyclase was first characterized by Zimmerman and Feng (28) as an enzyme which catalyzes the cyclization of 13-hydroperoxylinolenic acid to an 18-carbon fatty acid containing a cyclopentenone ring. The product, 8-[2-(cis-2'-pentenyl)-3-oxo-cis-4-cyclopentenylloctanoic acid, was given the common name 12-oxo-phytodienoic acid. Other fatty acid hydroperoxides can serve as substrates, but studies by us (24) showed that the hydro-peroxide must possess n-3 unsaturation to be a substrate for the cyclase enzyme. We have demonstrated the presence of the enzyme in a wide variety of plant tissues (23). The role of the products of these enzymes in plant metabolism is not known. In animal metabolism, however, in vitro experiments have shown that fatty acid hydroperoxides can accelerate the cyclooxygenase reaction, which is the initial step in the conversion of 20-carbon, polyunsaturated fatty acids to prostaglandins, thromboxanes, and prostacyclin (12). Activation of splenic cell guanylate cyclase by fatty acid hydroperoxides has also been demonstrated (7). Turner et al. (21) have provided evidence that 12-hydroxy-5,8,10,14-eicosatetraenoic acid, formed by enzymic reduction of the 12-hydroperoxy group, is a mediator of neutro-philic leukocyte chemotaxis. Whether these effects have in vivo significance has not been established. For a-or y-ketols of fatty acids and for 12-oxo-PDA2, no physiological role in either plants or animals has yet been demonstrated. Here, we report on the presence and properties oflipoxygenase, hydroperoxide isomerase, and hydroperoxide cyclase in young cotton seedlings. MATERIALS AND METHODS Chemicals. Linoleic and linolenic acids were obtained from Nu Chek Prep, Inc.3, Elysian, MN. Platinum oxide (Adam's catalyst) was purchased from Matheson, Coleman, and Bell, Norwood, OH, and methoxyamine-HCl in pyridine (MOX reagent) and N,O-bis(trimethylsilyl)-trifluoroacetamide were purchased from Pierce Chemical Co., Rockford, IL. Gas-Chrom Q (100/120 mesh) and the silicone phase DC LSX-3-0295 for GC were obtained from Applied Sciences, Inc., State College, PA. All TLC separations were accomplished with Anasil HF precoated TLC plates obtained from Analabs, Inc., New Haven, CT. Growth Conditions. Delinted cotton seeds (Gossypium hirsutum L. var. Stoneville 213) were soaked overnight in H20 without prior sterilization and then planted between two sheets of moist paper toweling 2 cm from the top. The moist towels were placed 2 Abbreviations: 12-oxo-PDA, 12-oxo-phytodienoic acid; TMS, tri-methylsilyloxy.