Identification and Characterization of Lipoxygenase Isoforms in Senescing Carnation Petals

Abstract

A membrane-associated lipoxygenase and a soluble lipoxygenase have been identified in camation (Dianthus caryophyllus L. cv Reve) petals. Treatments of microsomal membranes by nonionic or zwitterionic detergents indicated that lipoxygenase is tightly bound to the membranes. By phase separation in Triton X-114, microsomal lipoxygenase can be identified in part as an integral membrane protein. Soluble lipoxygenase had an optimum pH range of 4.9 to 5.8, whereas microsomal lipoxygenase exhibited maximum activity at pH 6.1. Both soluble and membraneassociated lipoxygenases produced carbonyl compounds and hydroperoxides simultaneously, in the presence of oxygen. The membranous enzyme was fully inhibited by 0.1 millimolar n-propyl gallate, nordihydroguaiaretic acid, or salicylhydroxamic acid, but the effect of the three inhibitors on the soluble enzyme was much lower. The soluble lipoxygenase is polymorphic and three isoforms greatly differing by their isoelectric points were identified. Lipoxygenase activity in flowers was maximal at the beginning of withering, both in the microsomal and the soluble fractions. Substantial variations in the ratio of the two forms of lipoxygenase were noted at different sampling dates. Our results allowed us to formulate the hypothesis of a strong association of one soluble form with defined membrane constituents.Lipoxygenases (EC 1. 13.11.12), commonly found in higher plants, catalyze the hydroperoxidation of polyunsaturated lipids to various primary and secondary oxidation products. Among characterized isoforms, similarities at the molecular level, high homologies, and cross-reactivities have been reported, suggesting a common evolutionary pathway of lipoxygenases in plants and animals (7). However, different isoforms of lipoxygenases can be distinguished on the basis of their reaction pH optima, protein isoelectric points, positional specificity of catalysis, or aerobic production of secondary products.Fat-oxidizing factors, like lipoxygenase, have been shown to be involved in processes linked to aging in various animal tissues and in processed vegetable products (14). Current theories on the physiological roles of plant lipoxygenases suggest involvement in such degradative processes as senescence, wounding, and infection, all of which include membrane breakdown (6, 16). Linoleic and linolenic acids, which are the principal substrates for lipoxygenases, are also major constituents of the phospholipid plant membrane component (14). Many plant lipoxygenases clearly show little reactivity toward esterified fatty acids, such as triglycerides and phospholipids (6). The release of free fatty acids catalyzed by phospholipases thus appears to be a probable prerequisite to the action of most plant lipoxygenases. Until recently, very little was known about the subcellular localization of the enzyme. Nonspecific absorption of lipoxygenase to membranous fractions was supposed to explain the difficulties encountered in the localization studies (16). However, the main cytosolic location of...

Lipid and oxylipin profiles during aging and sprout development in potato tubers (Solanum tuberosum L.)

“…Even though LOX is often proposed to be responsible for peroxidative damage to membrane lipids during aging and senescence, its role is far from clear. LOX activity increases during senescence of rose petals [17], daylily petals [18], carnation petals [19], and pea foliage [20], but clearly decreases during soybean seed aging [21], soybean cotyledon senescence [22] or in detached wheat and rye leaves [23]. In a previous study [24], we showed that in potato tubers stored at 20 jC, LOX activity and the total HPO content decreased during aging, even during the period after 120 days when membrane integrity dropped drastically.…”

Lipoxygenase activity associated to isolated soybean plasma membranes

“…Besides its cytosolic localization, LOX have been detected in different types of cellular membranes [11][12][13][14][15][16][17][18][19][20] and, in some cases, appear to be tightly bound to reconstituted membranes [12,14,[17][18][19][20], or present in spe cific organelles (chloroplasts or mitochondria) [12][13][14][15][16]. In particular, membrane-bound LOX, found in microsomal vesicles from tomato fruit and senescing carnation petals have been partially characterized [18,19]' Both utilize linoleic acid as substrate, have a pH optimum near to 6.0 and are distinguishable from their soluble counterpart. Membrane fractionation studies indicate that the tomato fruit enzyme is associated with thylakoid membranes [17].…”

“…However, they have been shown in vacuoles [11], mitochondria [12][13][14][15], chloroplasts [12,[15][16][17] and microsomal membranes [12,13,[18][19][20]. Hitherto, their presence in the plasmalemma of plant cells has not been conclusively demonstrated, albeit in a recent paper a plasma membrane-associated LOX has been de scribed [21].…”

Efeito da aplicação foliar de ácidos graxos na "Via das Lipoxigenases" de plantas de soja

“…No sistema vegetal, as lipoxigenases ocorrem em várias partes da planta, desempenhando funções em processos tais como: crescimento e desenvolvimento 1 , senescência 3 , reserva vegetativa 4 , germinação de sementes 5 , resposta a ferimento 6 e resistência a insetos e patógenos 7 . Os principais substratos de lipoxigenases, em plantas superiores, são os ácidos linoléico e linolênico 8 .…”