Lipoxygenase H1 Gene Silencing Reveals a Specific Role in Supplying Fatty Acid Hydroperoxides for Aliphatic Aldehyde Production

Abstract

Lipoxygenases catalyze the formation of fatty acid hydroperoxide precursors of an array of compounds involved in the regulation of plant development and responses to stress. To elucidate the function of the potato 13-lipoxygenase H1 (LOX H1), we have generated transgenic potato plants with reduced expression of the LOX H1 gene as a consequence of co-suppression-mediated gene silencing. Three independent LOX H1-silenced transgenic lines were obtained, having less than 1% of the LOX H1 protein present in wild-type plants. This depletion of LOX H1 has no effect on the basal or wound-induced levels of jasmonates derived from 13-hydroperoxylinolenic acid. However, LOX H1 depletion results in a marked reduction in the production of volatile aliphatic C6 aldehydes. These compounds are involved in plant defense responses, acting as either signaling molecules for wound-induced gene expression or as antimicrobial substances. LOX H1 protein was localized to the chloroplast and the protein, expressed in Escherichia coli, showed activity toward unesterified linoleic and linolenic acids and plastidic phosphatidylglycerol. The results demonstrate that LOX H1 is a specific isoform involved in the generation of volatile defense and signaling compounds through the HPL branch of the octadecanoid pathway. Lipoxygenase (LOX)1 enzymes catalyze the stereospecific dioxygenation of unsaturated fatty acids with a 1,4-pentadiene system. C18 unsaturated fatty acids, linoleic acid (18:2 ⌬9,12 ) and linolenic acid (18:3 ⌬9,12,15 ), are major LOX substrates in plants. The lipoxygenase pathway of fatty acid metabolism (1) is initiated by the addition of molecular oxygen at the C9 or C13 position of the acyl chain yielding the corresponding 9-and 13-hydroperoxides (2). Both 9-and 13-hydroperoxides can subsequently be cleaved to short-chain oxoacids and aldehydes by the action of hydroperoxide lyases (HPL) or, alternatively, the 13-hydroperoxide is converted, after enzymatic cyclization, reduction, and ␤-oxidation, to JA (3). Plant LOXs are ubiquitous and encoded by multigene families (4). The presence in a given tissue of several LOX isoforms with different substrate preferences, kinetic parameters, stereospecificity in substrate oxygenation, pH dependence, and subcellular localization makes difficult the assignment of specific functions to each LOX isoform. Moreover, LOX expression in plants is regulated throughout development and in response to stress (5, 6). Different LOX isoforms may have different physiological roles; they may, on the one hand, be responsible for the production of signals involved in the regulation of plant growth and the activation of stress-induced defense responses, whereas, on the other, the products of LOX activity may exert a direct deterring function toward pests and/or pathogens. It has been proposed that some aldehydes, in particular hexanal and hexenals produced by the action of HPL on LOX-derived fatty acid hydroperoxides, may be involved in the interaction between plants and pathogens or parasites (7-10) and, m...

Identification of a Specific Isoform of Tomato Lipoxygenase (TomloxC) Involved in the Generation of Fatty Acid-Derived Flavor Compounds

“…Lipoxygenases are also involved in JA synthesis, in generation of volatiles that attract some insects or insect predators, and in defense responses against insect pests. For example, depletion of potato LOX H3 reduced wound induction of proteinase inhibitors and increased weight gain of insect pests (Leon et al, 2002). This suggests that our range of transgenic plants with altered levels of different LOX isoforms (Fig.…”

“…Transcripts of LoxH1 are systemically up-regulated in both wounded and nondamaged tissues, indicating a role in signaling or defense (Royo et al, 1996). However, gene silencing of LoxH1 had no effect on the basal or wound-induced levels of jasmonates derived from 13-hydroperoxylinolenic acid (Leon et al, 2002). Potato plants with silenced LOX H3, a TomloxD homolog, showed greatly reduced proteinase inhibitor expression, indicating a role in defense signaling (Royo et al, 1999).…”

Variations in CYP74B2 (Hydroperoxide Lyase) Gene Expression Differentially Affect Hexenal Signaling in the Columbia and Landsberg erecta Ecotypes of Arabidopsis

“…Representing three of eight LOX loci in the Arabidopsis genome, these data suggest the LOX proteins encoded by these three genes generate substrates for the HPL branch cleaving 13-HPs and/or that they are activated by the products of a functional HPL pathway. Other LOX loci whose expression patterns do not vary between these ecotypes would be predicted to code for proteins generating substrates for the AOS branch of this pathway and/or metabolize other fatty acid HPs (Leó n et al, 2002;Halitschke and Baldwin, 2003). Underexpression of the LOX2 locus in the Col ecotype, which is induced by both insect damage and JA treatment in other ecotypes (Reymond et al, 2004) and implicated in generating precursors for both branches of the oxylipin pathway, is an especially important example of the autoregulatory cycles operating in this signaling pathway.…”

The Arabidopsis her1 mutant implicates GABA in E‐2‐hexenal responsiveness

“…In tobacco plants, in which HPL is silenced, the methyl jasmonate-induced proteinase inhibitor activity is strongly reduced, indicating that C6-volatiles, beside jasmonates, are required for this response (Halitschke et al, 2004). Similarly, Solanum tuberosum plants with reduced C6-volatile production show reduced induction of proteinase inhibitor (PIN2) expression upon wounding (Leó n et al, 2002). Furthermore, Arabidopsis plants, genetically modified to enhance C6-volatiles production, are less susceptible to infection with Botrytis cinerea, most likely as a result of both the activation of defense responses and the inhibition of fungal growth (Shiojiri et al, 2006a).…”