Cytochrome P450-dependent metabolism of oxylipins in tomato. Cloning and expression of allene oxide synthase and fatty acid hydroperoxide lyase

Abstract

Allene oxide synthase (AOS) and fatty acid hydroperoxide lyase (HPL) are plant-specific cytochrome P450s that commit fatty acid hydroperoxides to different branches of oxylipin metabolism. Here we report the cloning and characterization of AOS (LeAOS) and HPL (LeHPL) cDNAs from tomato (Lycopersicon esculentum). Functional expression of the cDNAs in Escherichia coli showed that LeAOS and LeHPL encode enzymes that metabolize 13- but not 9-hydroperoxide derivatives of C(18) fatty acids. LeAOS was active against both 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13-HPOT) and 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, whereas LeHPL showed a strong preference for 13-HPOT. These results suggest a role for LeAOS and LeHPL in the metabolism of 13-HPOT to jasmonic acid and hexenal/traumatin, respectively. LeAOS expression was detected in all organs of the plant. In contrast, LeHPL expression was predominant in leaves and flowers. Damage inflicted to leaves by chewing insect larvae led to an increase in the local and systemic expression of both genes, with LeAOS showing the strongest induction. Wound-induced expression of LeAOS also occurred in the def-1 mutant that is deficient in octadecanoid-based signaling of defensive proteinase inhibitor genes. These results demonstrate that tomato uses genetically distinct signaling pathways for the regulation of different classes of wound responsive genes.

Figure 1

Cyt P450-dependent metabolism of 13-HPOT. AOS (CYP74A) commits 13-HPOT to the production of JA and related cyclopenta(e) nones. In the absence of allene oxide cyclase (AOC), the epoxide product of AOS undergoes spontaneous hydrolysis to α- and γ-ketols and racemic 12-OPDA. HPL (CYP74B) cleaves 13-HPOT to produce C₆ and C₁₂ products that are further metabolized as shown.

Figure 2

Comparison of cDNA-deduced protein sequences of plant AOS and HPL genes. LeAOS and LeHPL sequences were aligned, using the ClustalW 1.7 program available at http://mbcr.bcm.tmc.edu/searchlauncher. AOS sequences were from flax (Song et al., 1993; accession no. U00428), guayule (Pan et al., 1995; accession no. X78166), and Arabidopsis (Laudert et al., 1996; accession no. Y12636). HPL sequences were from bell pepper (Matsui et al., 1996; accession no. U51674) and Arabidopsis (Bate et al., 1998; accession no. AF087932). Black boxes indicate amino acid residues that are conserved between all seven CYP74 members. Subfamily-specific substitutions are indicated with an asterisk. The three subfamily-specific motifs discussed in the text are underlined by the black bars. The symbol denotes the T → (I/V) change within the I helix that is a hallmark of CYP74 enzymes. The conserved Cys within the heme-binding domain is marked by a # symbol. The boxed residue (Pro-43) at the N terminus of LeAOS denotes the site where the His-tag was added in the pQE-AOS expression construct.

Figure 3

Southern-blot analysis of LeAOS and LeHPL. Genomic DNA from tomato was digested with restriction enzymes BamHI (B), EcoRV (E), XbaI (X), or BglII (Bg). DNA blots were hybridized to labeled probes derived from the open reading frame of LeAOS (left), LeHPL (middle), or the LeHPL 5′-UTR (right). Blots were hybridized in 5× SSPE at 65°C and washed in 0.5× SSPE at the same temperature, as described in “Materials and Methods.” Molecular mass standards (in kb) are indicated on the left.

Figure 4

Activity of LeAOS and LeHPL expressed in E. coli. Total lysates of E. coli cells expressing pQE-AOS, pQE-HPL, or the empty vector (pQE-30) were tested for their ability to metabolize C₁₈ (13-HPOD and 13-HPOT) and C₂₀ (15-HPET) fatty acid hydroperoxides. Activity was measured either directly as a decrease in absorbance of the substrate at A₂₃₄ (A) or indirectly as the production of aldehydes using a NADH-coupled assay (B). Error bars represent the mean and sd of activity determined from three enzyme preparations of each culture.

Figure 5

Expression of LeAOS and LeHPL genes in different organs of tomato. Total RNA was extracted from roots (R), stems (S), leaves (L), developing flower buds (B), mature unopened flowers (UF), mature opened flowers (OF), small (<0.5 cm) immature green fruit (IF), mature green fruit (GF), or mature red fruit (RF). Ten-microgram samples of RNA were subjected to RNA-blot analysis. Specific transcripts were detected by hybridization of blots to probes corresponding to full-length LeAOS, full-length LeHPL, the 5′-UTR of LeHPL, or an eIF4A probe used as a loading control. Also shown is a photograph of an ethidium bromide-stained gel of the RNA used for the experiment (EtBr).

Figure 6

Accumulation of LeAOS protein in different organs of tomato. Fifteen-microgram samples of membrane protein prepared from young flower buds (buds), roots (root), stems (stem), petioles (pet), cotyledons (cot), and leaves (leaf) were separated by SDS-PAGE. Protein was transferred to Immobilon-P membranes and probed with either antiserum raised against LeAOS (left) or an equivalent amount of preimmune serum (right). The numbers on the left of the figure indicate the position of M_r standards.

Figure 7

Accumulation of LeAOS, LeHPL, and Inh-II mRNAs in tomato plants in response to herbivory. Tobacco hornworm larvae (third instar) were placed onto the lower leaf of 3-week-old cv Micro-Tom plants and allowed to feed for 5 to 10 min. During this period, approximately 5% to 10% of the area of the attacked leaf was consumed by the larvae. Leaf tissue was harvested for extraction of total RNA immediately after removal of the larvae (0 point) or at the times indicated (in h). RNA was prepared separately from the lower damaged leaf (Local response) and from the third leaf (counted from the base of the plant) (Systemic response). RNA was also prepared from a set of control plants that received no damage (C). Duplicate RNA blots containing 5 μg of RNA per sample were hybridized to cDNA probes for proteinase inhibitor II (Inh-II), LeAOS, LeHPL, and eIF4A as a loading control.

Figure 8

Analysis of wound-induced gene expression in wild-type and def-1 mutant plants. Fifteen-day-old wild-type (cv Castlemart) and def-1 mutant seedlings were mechanically wounded at the distal end of the terminal leaflet of the lower leaf. Undamaged tissue on the same leaflet was harvested for RNA extraction at the indicated times after wounding. RNA blots were hybridized to cDNA probes for proteinase inhibitor II (Inh-II), cathepsin D inhibitor (CDI), TomLoxD (LoxD), LeAOS (AOS), LE RNase (LE), and eIF4A as a loading control.