Soybean Leaves Contain Multiple Lipoxygenases

Abstract

Chromatofocusing of soybean (Glycine max L.) leaf lipoxygenases revealed three distinct peaks of activity. Based on their isoelectric points (pis), pH optima, and mutant analysis it appears that the leaf isozymes are different from those described from mature soybean seed. At least one leaf lipoxygenase appears to differ from those found in hypocotyls. The pis of the main bands of the three leaf lipoxygenase peaks are 6.67, 5.91, and 5.67. The pH optima curves of three active fractions exhibit peaks at pH 6.2, 5.5, and 8.5, respectively. One of the fractions has two polypeptides with slightly different molecular weights, both of which react to soybean seed lipoxygenase antibodies. The other two fractions contain a polypeptide of unit molecular weight reacting with the lipoxygenase antibodies.LOXs2 (linoleate: oxygen oxidoreductase, EC 1. 13.11.12) are found throughout the plant (8) and animal kingdoms (9). They catalyze, in plants, the hydroperoxidation of cis,cis-1,4-pentadiene structures present in unsaturated fatty acids. The physiological role of LOX in plants remains ambiguous, although roles in growth and development, senescence or wound response, and pest resistance have been suggested ( 1 3, 15, 19).While soybean seed LOX has been extensively studied with three different isozymes described (2,12) sunlight. Leaves were measured at the maximum width, weighed, and ground in one-half volume of water with a mortar and pestle. Samples were centrifuged at 12,000g for 15 min and the supernatants were analyzed for LOX activity (11). Enzyme PurificationThe middle leaflets of soybean (Glycine max L. Merr.) trifoliates were harvested at random from plants growing in a greenhouse, frozen in liquid nitrogen, and ground with a mortar and pestle. Following grinding, 5 mM Na acetate (pH 4.5) was added (80 mL per 20 leaflets) and the resulting slurry was centrifuged for 7.5 min at 10,000g. The supernatant was made 10% in sucrose and loaded onto a Sephadex G-50 (coarse, 55 x 5 cm) (Pharmacia) column and eluted in 5 mM Na acetate, pH 4.5 at about 8 mL min-'. The protein containing fractions were analyzed for LOX activity by measuring the change in absorbance at 234 nm with time (11). Sodium linoleate was used as the LOX substrate. This was adjusted to an absorbance of 0.45 with previously prepared (partially oxidized) substrate. The most active fractions were pooled and centrifuged for 1.5 h at 140,000g to remove microsomal particles. The resulting supernatant was loaded onto a PBE94 chromatofocusing column (Pharmacia) previously equilibrated to pH 7.4 with 25 mM imidazole HCl solution. The proteins were eluted using Polybuffer 74 (Pharmacia), which had been diluted 1 to 8 and adjusted to pH 4.0 with 1 M HCl.Every other fraction collected from the chromatofocusing column was analyzed for LOX activity, and the approximate pls of the LOX proteins were determined by comparison to pI standards (Bio-Rad) on IEF-PAGE gels (see below). The samples with the highest LOX activity in a single peak were pooled and concentrated ...

Effect of Volatile Methyl Jasmonate on the Oxylipin Pathway in Tobacco, Cucumber, and Arabidopsis

“…Western blotting was performed as described by Wang and Hildebrand (1987) using rabbit polyclonal antibodies raised against soybean leaf peak 3 LOX (Grayburn et al, 1991). Fifty micrograms of the protein extracted from tobacco leaves were analyzed on a 10% SDS gel and probed with polyclonal antibodies raised against soybean leaf peak 3 LOX isozyme.…”

The Lipoxygenase Isozymes in Soybean [Glycine max (L.) Merr.] Leaves (Changes during Leaf Development, after Wounding, and following Reproductive Sink Removal)

“…Vol. 107, 1995 In soybean leaves, total lipoxygenase protein and activity have been reported to vary with respect to leaf size (Hildebrand et al, 1988;Altschuler et al, 1989;Grayburn et al, 1991). Because soybean leaves contain multiple lipoxygenase isozymes, variation in total lipoxygenase protein and activity during Ieaf development probably results from changes in individual lipoxygenase isozymes, but the relationship of individual lipoxygenase isozyme levels to leaf age has not been established.…”

“…Multiple lipoxygenase isozymes also occur in soybean leaves. Grayburn et al (1991) used chromatofocusing to separate three distinct peaks of lipoxygenase activity in extracts from soybean leaves. On the basis of physical and kinetic characterizations of the three leaf lipoxygenases, Grayburn et al (1991) concluded that the soybean leaf lipoxygenase isozymes are distinct from the isozymes found in seeds and that at least one of the leaf isozymes is different from the lipoxygenase isozymes found in hypocotyl/radicle axes.…”

“…Grayburn et al (1991) used chromatofocusing to separate three distinct peaks of lipoxygenase activity in extracts from soybean leaves. On the basis of physical and kinetic characterizations of the three leaf lipoxygenases, Grayburn et al (1991) concluded that the soybean leaf lipoxygenase isozymes are distinct from the isozymes found in seeds and that at least one of the leaf isozymes is different from the lipoxygenase isozymes found in hypocotyl/radicle axes. IEF followed by immunoblotting has been used to demonstrate that each of the three leaf lipoxygenase activity peaks separated by chromatofocusing contains severa1 distinct lipoxygenase isoforms (Grayburn et al, 1991).…”

Biochemical Constituents Imparting Resistance to Sucking Pest Aphid in Cotton (Gossypium spp.)

“…Lipoxygenase activity in the leaves was determined colorimetrically (Grayburn et al, 1991). 29.9ml of linoleic acid was taken in 100ml volumetric flask and maintained at 30 0 C using hot water bath.…”