cDNA cloning of rice lipoxygenase L‐2 and characterization using an active enzyme expressed from the cDNA in Escherichia coli

Abstract

A full-length cDNA of rice lipoxygenase L-2 was cloned from 3-day-old seedlings. The identity of the clone was determined by amino acid sequencing of selected peptides of the purified enzyme and immunological characterization of an active enzyme that was produced from the cDNA in Escherichia coli by cultivation at 15°C. The nucleotide sequence showed a strong bias toward G and C in the selection of nucleotides, especially at the third position of the codons (93% G/C). The complete amino acid sequence of the enzyme was deduced from the nucleotide sequence. The molecular mass of the enzyme was calculated to be 96657 Da based on 865 amino acids. The amino acid sequence shares similarity with those of dicot lipoxygenases throughout the enzyme at a level of 50%. A hydropathy profile calculated from the amino acid sequence resembled those of dicot lipoxygenases, suggesting conservation of the secondary structure of these enzymes. The active enzyme, expressed in Escherichia coli, was characterized for pH dependence of the enzyme activity, intramolecular specificity, heat stability and K,. The enzyme had the same properties as the L-2 enzyme that was isolated from seedlings, but differed from the lipoxygenase L-3 isolated from mature plants.Lipoxygenase catalyzes hydroperoxidation of fatty acids by the introduction of molecular oxygen onto the cis, cis-1,4-pentadiene structure of its substrates. The hydroperoxy fatty acids are then metabolized into physiologically active compounds. Eicosanoids, which are derived from arachidonic acid via lipoxygenation pathways in mammalian cells have been extensively investigated [ 11. Jasmonic acid, derived from linolenic acid via lipoxygenation in plant cells, is recognized as a gaseous growth regulator [2]. Extensive enzyme studies of lipoxygenases, especially soybean lipoxygenase L-1 , have been carried out [3] and two recent reviews of plant lipoxygenases have been published [4, 51. Recently, biosynthesis of anti-fungal compounds via a lipoxygenase pathway was proposed by Ohta et al. [6]. The same authors also demonstrated an increase in lipoxygenase activity in rice leaves in response to infection with an incompatible strain of blast fungus [7] (a compatible strain did not elicit increased lipoxygenase activity), suggesting an important role for lipoxygenase in plant disease resistance.Characterization of rice lipoxygenases in mature seeds has been reported [8 -101. The seeds contain three distinct lipoxygenases, L-1, L-2 and L-3. They differ from each other not only in their enzymic properties, but also in their physiological aspects. The L-3 enzyme is the major lipoxygenase in Correspondence to D. Shibata, Mitsui Plant Biotechnology Research Institute, TCI-AIO, Sengen 2-1-6, Tsukuba, Ibaraki, Japan 305Abbreviation. L-E, an active enzyme expressed from the full-length cDNA encoding a rice lipoxygenase in E. coli.Enzyme. Lipoxygenase (EC 1.13.11.12).Note. The nucleotide-sequence data published here have been submitted to the EMBL sequence data bank and are available under acces...

The rice <i>GRH2</i> and <i>GRH4</i> activate various defense responses to the green rice leafhopper and confer strong insect resistance

“…In contrast, the expression of OsLOX-RLL (Os08g39840) was downregulated in response to GRH infestation in TGRH29. These transcription of these LOX genes was differentially regulated by the GRH2/ GRH4-mediated defense response, although OsRCI-1 and OsLOX-RLL encode 13-LOX, which incorporates oxygen at the C-13 position of the fatty acid to yield a 13-fatty acid hydroperoxide (Ohta et al 1992, Peng et al 1994, Schaffrath et al 2000. The expression of OsRCI-1 is induced in response to treatment with 2,6-dichloroisonicotinic acid (INA), JA, BTH and probenazol (Schaffrath et al 2000).…”

Mechanosensitive Expression of a Lipoxygenase Gene in Wheat

“…1). The amino acid sequence of the WCI-2-encoded protein is 64% identical and 78% similar to LOX L-2 from rice (Ohta et al, 1992) and 60% identical and 74% similar to the LOX2 sequence of soybean (Shibata et al, 1987). The His residues conserved in mammalian and plant LOXs (Siedow, 1991) are also conserved in the WCI-2 cDNA sequence.…”

The Cloning of Two Tomato Lipoxygenase Genes and Their Differential Expression during Fruit Ripening

“…There is growing evidence that plant lipoxygenases are involved in several physiological functions including growth and development (Kato et al, 1992;Matsui et al, 1992;Ohta et al, 1992), defense against wounding and pathogens (Bell and Mullet, 1991;Croft et al, 1993;Foumier et al, 1993;Melan et al, 1993), and senescence (reviewed by Paliyath and Droillard, 1992). Senescence and fruit ripening are characterized by an early deterioration of cellular membranes.…”