ALLENE OXIDE SYNTHASE and HYDROPEROXIDE LYASE, Two Non-Canonical Cytochrome P450s in Arabidopsis thaliana and Their Different Roles in Plant Defense

Abstract

The channeling of metabolites is an essential step of metabolic regulation in all living organisms. Multifunctional enzymes with defined domains for metabolite compartmentalization are rare, but in many cases, larger assemblies forming multimeric protein complexes operate in defined metabolic shunts. In Arabidopsis thaliana, a multimeric complex was discovered that contains a 13-lipoxygenase and allene oxide synthase (AOS) as well as allene oxide cyclase. All three plant enzymes are localized in chloroplasts, contributing to the biosynthesis of jasmonic acid (JA). JA and its derivatives act as ubiquitous plant defense regulators in responses to both biotic and abiotic stresses. AOS belongs to the superfamily of cytochrome P450 enzymes and is named CYP74A. Another CYP450 in chloroplasts, hydroperoxide lyase (HPL, CYP74B), competes with AOS for the common substrate. The products of the HPL reaction are green leaf volatiles that are involved in the deterrence of insect pests. Both enzymes represent non-canonical CYP450 family members, as they do not depend on O₂ and NADPH-dependent CYP450 reductase activities. AOS and HPL activities are crucial for plants to respond to different biotic foes. In this mini-review, we aim to summarize how plants make use of the LOX2-AOS-AOC2 complex in chloroplasts to boost JA biosynthesis over volatile production and how this situation may change in plant communities during mass ingestion by insect pests.

Keywords: allene oxide cyclase; allene oxide synthase; chloroplast envelope protein complex; hydroperoxide lyase; lipoxygenase; metabolite channeling; plant defense.

Figure 1

Biosynthesis of jasmonic acid (JA) and green leaf volatiles through the Vick and Zimmerman shunt. Pathway intermediates are given as 13-HPOT, (9Z11E15Z13S)-13-hydroperoxy-9,11,15-octadecatrienoic acid; α-LeA, α-linolenic acid; EOT, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid; OPDA, cis-(+)-12-oxophytodienoic acid; ODA, 12-oxo-cis-9-dodecenoic acid. Enzymes abbreviations refer to LOX, 13-lipoxygenase; AOS, 13-allene oxide synthase; AOC, allene oxide cyclase; HPL, 13-hydroperoxide lyase. Note that 13-HPOT is a common substrate of AOS and HPL that, as non-canonical CYP450 enzymes, drive alternative reactions in the JA and green leaf volatile branches of the Vick and Zimmerman pathway.

Figure 2

Structural models of the 8R-LOX of Plexaura homomella and LOX2 of Arabidopsis. A homology model of AtLOX2 was established with soybean LOX3 (PDB ID: 1LNH) as template [65] using SWISS-MODEL [66] and, in turn, super-positioned with the structure of the crystallized 8R-LOX domain (PDB ID: 2FNQ) of the soft coral 8R–LOX–AOS [59]. To overlay the structures, the Smith–Waterman superposition algorithm on the Calculate Structure Alignment.app was used. (a) 3D structure of LOX2 (mustard color) and the 8R-LOX domain (teal color) of the soft coral 8R–LOX–AOS. (b) Amino acid sequence alignment to highlight the conservation of structural domains and active site residues. Active site residues and iron binding sites (boxed) are indicated [62]. Transmembrane domain prediction was done using TMpred [67].

Figure 3

Structural model of the LOX2–AOS–AOC2 complex in chloroplasts. (a) Overall structure that was obtained by fitting the modelled LOX2 structures of Arabidopsis to the known X-ray structures of AOS [44] and AOC2 [32]. Amino acid residues suggested to be involved in catalysis of each enzyme as well as amino acid residues tentatively defined as residues mediating subunit interactions are indicated. (b) Cartoon highlighting the different amino acid pairs implicated in the different protein–protein interactions.

Figure 4

Tissue and development-specific expression of AOC1-4 from Arabidopsis thaliana. The Genevestigator database [74] was used to contrast the specific expression pattern of AOC1-4. The patterns reveal a clear expression of AOC1 and 2 in areal tissues, while being absent in roots and only very little expressed in flowers. AOC4 is seemingly restricted to root tissues, whereas AOC3 expression is detected at different levels in all tissues.

Figure 5

Compartmentalization of the AOS and HPL branches of the Vick and Zimmerman pathway in plants. (a) Under unperturbed conditions, the link between LOX2 and AOS is less strong, causing a portion of 13-HPOT to leak out of the enzymatic channel. The free 13-HPOT serves as substrate for HPL to produce green leaf volatiles. (b) In response to herbivory, however, the amount of chloroplastic AOS and AOC increases, effectively recruiting LOX2 into the LOX2–AOS–AOC complex. This prevents leakage of 13-HPOT and consequently promotes cis-(+)-OPDA production.

Figure 6

Reaction mechanism proposed on the basis of the bifunctional 8R–LOX–AOS and 8R–LOX–HPL enzymes from cyanobacteria and Capsella imbricate. Modified after Teder et al. [79].