Purification and characterization of elicitor‐induced lipoxygenase in tobacco cells

Abstract

SummaryLipoxygenase activity was induced in a tobacco cell suspension culture by treatment with glycopeptide elicitors prepared from the cell walls of Phytophthora parasitica var, nicotianae, and in tobacco seedlings infected by this fungal pathogen. Upon purification and characterization, the enzyme appeared to have a molecular weight of 96000, a pl of 5.1 and a Km of 20.9 μM with linoleic acid as substrate. According to its acidic optimum pH, it belongs to type‐2 lipoxygenases. Using linoleic, linolenic and arachidonic acids as substrates, the products formed in vitro by lipoxygenase were characterized. 9‐ and 5‐hydroperoxides were the main products obtained from the C18 and C20 fatty acids, respectively, thereby indicating that a 5‐lipoxygenase accounts for most of the elicitor‐induced activity, since the main site of insertion of molecular oxygen is on C‐5 of arachidonic acid. Small amounts of 13‐hydroperoxides were also formed from the C18 fatty acids. In vitro, the strongest inhibitors of tobacco lipoxygenase were n‐propylgallate and nordihydroguaiaretic acid. The possible involvement of this enzyme in signaling phenomena leading to defense induction in plants via jasmonic acid and other fatty acid‐derived products is discussed.

Role of Jasmonates in the Elicitor- and Wound-Inducible Expression of Defense Genes in Parsley and Transgenic Tobacco

“…nPG strongly inhibits the activity of a tobacco lipoxygenase that may be involved in JA biosynthesis from LA (Fournier et al, 1993) and has been shown to inhibit wound-induced expression of pin and vsp genes (Staswick et al, 1991;PeÍía-Cortes et al, 1993). In parsley cells nPG was partially effective in reducing the elicitorinducible expression of 4CL, PAL, and TyrDC but was ineffective in reducing EL13 or HRGP expression.…”

“…The ability of nPG to effectively inhibit plant oxygenases and thus biosynthesis of jasmonates via the octadecanoid pathway is related to its activity as an antioxidant, free radical scavenger (Staswick et al, 1991;Fournier et al, 1993). Activated oxygen species are produced following elicitor treatment of parsley cells (Niirnberger et al, 1994), and such species could play a role in elicitor-induced signal transduction (Aposto1 et al, 1989;Devlin and Gustine, 1992;Mehdy, 1994).…”

“…The free radical scavenger nPG is a well-characterized lipoxygenase inhibitor. Because of its potency in inhibiting tobacco lipoxygenase (Fournier et al, 1993), an activity required for the de novo biosynthesis of JA from LA, we tested the ability of this compound to inhibit elicitor-activated gene expression in parsley cells and its ability to inhibit wound-activated gene expression in tobacco.…”

Characterization of a Divinyl Ether Biosynthetic Pathway Specifically Associated with Pathogenesis in Tobacco

“…In tobacco, 9-hydroperoxides are expected to be formed by NtLOX1, an elicitor-and pathogen-inducible 9-LOX (Fournier et al, 1993;Véronési et al, 1996). Therefore, the ability of NtDES1 to utilize hydroperoxides formed through NtLOX1 action was investigated in a qualitative thin-layer chromatography (TLC) assay.…”

“…Thus, 9-LOX gene expression and 9-LOX activity are strongly induced in tobacco and potato in response to two oomycete pathogens of these plants, Phytophthora parasitica var. nicotianae (Ppn) and Phytophthora infestans, respectively, and to oomycete-derived elicitors (Fournier et al, 1993;Véronési et al, 1996;Fidantsef and Bostock, 1998;Kolomiets et al, 2000;Gö bel et al, 2001). Similarly, expression of the 9-DES gene StDES is increased in potato in response to elicitors and pathogen attack (Stumpe et al, 2001).…”