Molecular Characterization of an Arabidopsis Gene Encoding Hydroperoxide Lyase, a Cytochrome P-450 That Is Wound Inducible1

Abstract

Hydroperoxide lyase (HPL) cleaves lipid hydroperoxides to produce volatile flavor molecules and also potential signal molecules. We have characterized a gene from Arabidopsis that is homologous to a recently cloned HPL from green pepper (Capsicum annuum). The deduced protein sequence indicates that this gene encodes a cytochrome P-450 with a structure similar to that of allene oxide synthase. The gene was cloned into an expression vector and expressed in Escherichia coli to demonstrate HPL activity. Significant HPL activity was evident when 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid was used as the substrate, whereas activity with 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid was approximately 10-fold lower. Analysis of headspace volatiles by gas chromatography-mass spectrometry, after addition of the substrate to E. coli extracts expressing the protein, confirmed enzyme-activity data, since cis-3-hexenal was produced by the enzymatic activity of the encoded protein, whereas hexanal production was limited. Molecular characterization of this gene indicates that it is expressed at high levels in floral tissue and is wound inducible but, unlike allene oxide synthase, it is not induced by treatment with methyl jasmonate.In green plant tissue HPL cleaves C18-lipid hydroperoxides to form a C6-aldehyde and a 12-carbon oxoacid (Hatanaka, 1993). The C12 product of HPL leads to the formation of traumatin, which is implicated in wound signaling (Zimmerman and Coudron, 1979). The C6-aldehyde products of the HPL reaction depend on the substrate; cis-3-hexenal is formed from HPOT and hexanal is formed from HPOD. The lipid hydroperoxide substrates were generated from C18 free fatty acids by the enzymatic activity of lipoxygenase (see Fig. 1). HPL activity has been found in a variety of plants and is thought to be associated with the chloroplast envelope (Blée and Joyard, 1996), developmentally regulated (Vick and Zimmerman, 1976;Gardner et al., 1991;Riley et al., 1996), and discernible as two distinct activities in tea leaves (Matsui et al., 1991).Although the central lipoxygenase pathway is present in animal systems, and the formation of prostaglandins and leukotrienes in animals is analogous to the formation of jasmonates in plant tissue (Anderson, 1989), there is no pathway in animals analogous to the HPL branch pathway. The C6 compounds produced by HPL are released rapidly from disrupted plant tissue and form the basis for the "green note" flavor characteristic of plant tissue. The green note flavor is an important determinant of fresh fruit and vegetable quality, and C6 volatiles are widely used as a prepared food additive (Hatanaka, 1993). C6 volatiles produced from this pathway also have antimicrobial properties, suggesting that they may play a protective role in plant defense (Croft et al., 1993). They may also have an application as antimicrobial fumigants in postharvest storage (Archbold et al., 1997). In addition, C6 volatiles of the HPL pathway induce phytoalexin accumulation (Zeringue, 1992) and inhi...

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

“…4). In agreement with this conclusion, previous studies in which HPOD was incubated alone (Grosch, 1987) or in Escherichia coli extracts (Bate et al, 1998b) have also indicated that this compound readily autooxidizes to form hexanal.…”

“…Database analyses have indicated that there are six full-length CYP74B2 cDNA records having GenBank accession numbers AF087932 (Bate et al, 1998b), NM_117633 (reference sequence derived from an annotated genomic sequence NC_003075), BX827042, BX826974, BX826954, and BX826430; because the last four records are individual clones from a single cDNA library, they are considered as one source. Although records from all three sources report that they are derived from the Col ecotype, the last two sets of sequences (NM_117633 and four BX82XXXX) differ in the deletion of 10 nucleotides (nts; gray box in Fig.…”

“…2, gray box) to code for an N-terminal truncated protein containing 385 amino acids and an in-frame P450 signature sequence. Deleted from this second predicted protein, which is substantially shorter than all other known P450 proteins, is the entire transit sequence needed for chloroplast targeting (Bate et al, 1998b) as well as some N-terminal residues found within HPLs of different plant species (Froehlich et al, 2001). …”

“…P450 proteins within this family are unique when compared to most other P450 proteins in that they do not require molecular oxygen or utilize NADPH for dioxygen activation (Song and Brash, 1991;Lau et al, 1993;Shibata et al, 1995), a feature that has often caused them to be described as nonclassical P450 proteins. Also, in contrast to the endoplasmic reticulum localization of most other P450s, gene comparisons have revealed the existence of an N-terminal transit peptide appropriate for chloroplast targeting (Song et al, 1993;Laudert et al, 1996;Bate et al, 1998b). Subcellular fractionations in tomato (Lycopersicon esculentum) have indicated that the CYP74A member of this family in tomato is localized on the inner chloroplast membrane facing the stroma and the CYP74B member of this family is localized in the outer chloroplast membrane facing the intermembrane space (Froehlich et al, 2001); other studies simply localize members of this P450 family to the chloroplast without differentiating between its various subcompartments.…”

“…Subcellular fractionations in tomato (Lycopersicon esculentum) have indicated that the CYP74A member of this family in tomato is localized on the inner chloroplast membrane facing the stroma and the CYP74B member of this family is localized in the outer chloroplast membrane facing the intermembrane space (Froehlich et al, 2001); other studies simply localize members of this P450 family to the chloroplast without differentiating between its various subcompartments. Biochemically, each of these enzymes has a distinct function with CYP74A (allene oxide synthase [AOS]) members mediating the synthesis of 12-oxo-phytodienoic acid (12-OPDA), jasmonic acid (JA), and its methyl ester (MeJA) from fatty acid hydroperoxide (HP) in the lipoxygenase (LOX) pathway of oxylipin synthesis (Laudert et al, 1996;Laudert and Weiler, 1998;Kubigsteltig et al, 1999) and CYP74B (hydroperoxide lyase [HPL]) members mediating the breakdown of HP to C 6 volatiles and octadecanoic acid (Bate et al, 1998b;Matsui et al, 1999;Fig. 1).…”