Plants lack an immune system like animals. However, they possess mechanisms that recognize pathogens and initiate defense responses. Some of these mechanisms involve various types of oxygenated fatty acids, termed “oxylipins.” These molecules are involved in responses to physical damage by animals or insects, stress, and attack by pathogens. Oxylipins are derived from linoleic and α-linolenic acids, released from their lipid associations by poorly defined acyl hydrolases (lipases) of various kinds (Figure 1). A first key step in oxidation is the action of lipoxygenases. For example, depending on the source of the enzyme, lipoxygenases (e.g., LOX1, a 9-lipoxygenase from the cytosol, or LOX2, a 13-lipoxygenase from the chloroplast stroma) catalyze the oxidation of linoleic (C18:2) or linolenic acids (C18:3) into either 9- or 13-hydroperoxy-octadecatrienoic acids (HPODE). Such compounds are highly reactive, and they are quickly metabolized by various enzymes into series of oxylipins, with a range of distinct biological activities (Blée, 2002; Mosblech et al., 2009; Joyard et al., 2010). Some oxylipins such as 4-oxononenal, 4-hydroxynonenal, ketodienes [the 9-oxo-10(E), 12(E)-octadecadienoic acid (9-KODE), and 13-oxo-9(Z), 11(E)-octadecadienoic acid (13-KODE)], ketotrienes [the 9-oxo-10(E), 12(Z), 15(Z)-octadecatrienoic acid (9-KOTE) and 13-oxo-9(Z), 11(E), 15(Z)-octadecatrienoic acid (13-KOTE)], or the plant specific γ-ketols (Figure 1 and Figure S1) are α,β-unsaturated carbonyls and reactive electrophile species (RES). RES can act as signaling molecules (Farmer and Mueller, 2013) and also react with important cellular nucleophiles such as thiols of proteins and nucleic acids, modifying their biochemical properties, and are potentially toxic (Esterbauer et al., 1991; Szweda et al., 1993). Due to this potential toxicity, cells have developed an armory of defense to decrease their activity. One class of enzymes involved in detoxification of reactive electrophile oxylipins are NADPH-oxidoreductases belonging to the zinc-independent medium-chain dehydrogenase/reductase (MDR) superfamily and more specifically to the quinone oxidoreductase (QOR) subfamily to which alkenal/one reductases belong (Porté et al., 2009). These enzymes can decrease the activity of reactive electrophile oxylipins by reducing the unsaturated carbon-carbon bond located in α,β of the carbonyl group to a single bond (Yamauchi et al., 2011) (Figure 1).

The chloroplast envelope Quinone Oxidoreductase Homolog (ceQORH; At4g13010; molecular weight 34,034 Da) from Arabidopsis thaliana is associated to the inner membrane of the chloroplast envelope (Figure 1) where it represents 1–2% of the crude envelope proteins (Miras et al., 2002). It is encoded by the nuclear genome and is targeted to the chloroplast by an alternative import pathway, without cleavage of its internal chloroplast targeting sequence (Miras et al., 2002, 2007). Despite its original annotation as a “QOR,” ceQORH is inactive on quinones but preferentially reduces γ-ketols in the presence of NADPH (Curien et al., 2016) (Figure 1 and Figure S1). γ-ketols (Figure 1 and Figure S1) are long-chain reactive electrophile oxylipins and are potentially toxic (Kuga et al., 1993). They are spontaneously produced in the jasmonate biosynthetic pathway, downstream of lipoxygenase specific peroxidation by hydrolysis of an allene oxide intermediate (Grechkin et al., 1991). ceQORH is also active though to a lesser extent on the highly toxic (Lin et al., 2005) C9 α,β-unsaturated carbonyl 4-oxononenal (Curien et al., 2016). ceQORH is thus probably dedicated to detoxification of γ-ketols which can be produced in plants under normal growth conditions (Theodoulou et al., 2005), and accumulate in damaged tissues (Buseman et al., 2006) or at a distance from bite zone when plants are attacked by caterpillars (Schulze et al., 2007). ceQORH bears similar characteristics and sequence homology with members of the MDR superfamily, which includes chloroplastic alkenone reductase [At1g23740, AtAOR (Yamauchi et al., 2012), Figure 1], Arabidopsis ζ-crystallin [alkenal reductase, AT5G16970, AtAER (Mano et al., 2005), Figure 1], yeast ζ-crystallin (Crosas et al., 2011), human eye ζ-crystallin (Porté et al., 2011), and Escherichia coli quinone reductase (Thorn et al., 1995) (Figure S2).

Unlike the chloroplastic AtAOR (Yamauchi et al., 2012) (Figure 1), which is active on short-chain α,β-unsaturated carbonyls (C < 5), ceQORH does not reduce short chain unsaturated carbonyls (C < 9). In addition, compared to the cytosolic broad specific alkenal/one reductase AtAER (Figure 1), ceQORH showed a restricted substrate specificity, being inactive on the ketodienes 9-KODE and 13-KODE, or on the ketotrienes 9-KOTE and 13-KOTE (Figure S1) and virtually inactive on 4-hydroxynonenal and traumatin [12-oxo-10(E) dodecenoate] (Curien et al., 2016). ceQORH is encoded by the nuclear genome and is targeted to the chloroplast by an alternative import pathway independent from the trimeric TOC159/75/34 complex, without cleavage of its internal chloroplast targeting sequence (Miras et al., 2002, 2007). This peculiarity, together with the ceQORH restricted substrate specificity, prompted us to carry out crystallographic studies of ceQORH. No crystal was obtained either in the presence of γ-ketols or NADPH but the protein crystallized in the absence of ligands as well as bound to 13-KODE and NADPH, 13-KOTE and NADP⁺ and 13-KOTE alone. Structure comparisons with AtAER (Youn et al., 2006) and the enone oxidoreductase from Fragaria x ananassa (Schiefner et al., 2013) provided insights into the molecular basis of substrate specificity.

9,12 γ-ketols are plant specific reactive electrophile oxylipins produced spontaneously in chloroplasts (Grechkin et al., 1991). Until recently, no enzyme able to detoxify γ-ketol in the chloroplast could be identified. Previously, we showed that ceQORH, a chloroplast inner envelope membrane associated protein, efficiently reduces γ-ketol but is inactive with the ketodienes 13-KODE and 13-KOTE and small reactive aldehydes (less than 9 carbon atoms) (Curien et al., 2016). Structure analyses of ceQORH crystallized bound to ligands showed that the ligand binding site is large and solvent exposed. These features are consistent with the binding of medium-sized (C > 9) molecules such as 1,3-diphenyl-2-propenone or 4-oxononenal but also with the binding of the long-chain molecules 9,12 γ-ketols (18 carbon atoms) (Curien et al., 2016). The structure analysis of the ternary complex ceQORH-NADP⁺-13-KOTE provides a structural explanation for the absence of activity with 13-KOTE with a positioning inconsistent with a hydride transfer from NADPH to the C = C bond positioned in α,β of the ketone. This observation also suggests that positioning of γ-ketol in the ligand binding site of ceQORH is different from that observed for 13-KOTE, γ-ketol being necessarily closer from the C4 of NADPH to be reduced. Structure comparisons and kinetic studies revealed that ceQORH shares structural similarities and overlapping substrate specificity with the cytoplasmic NADPH-dependent 2-alkenal reductase (AtAER) from A. thaliana (PDB entries: 2J3J and 2J3K) (Mano et al., 2005; Youn et al., 2006). AtAER is able to reduce the same substrates as ceQORH with similar efficiencies. However, AtAER substrate specificity is much larger than that of ceQORH, being active on ketodiene and ketotriene and showing much higher affinity for 4-hydroxy-2-nonenal and the C12 molecule traumatin (Curien et al., 2016). The structure comparison shows that both enzymes have a large ligand binding site. More generally, structure comparison with QORs and sequence alignments showed that the residues of ceQORH binding site are not well-conserved. Only co-crystallization of ceQORH and other QORs with substrates would allow understanding the basis of ceQORH restricted substrate specificity. Unfortunately we could not obtain crystals in the presence of γ-ketols. Nonetheless, by comparing different structures of QORs we could highlight a molecular basis allowing selection of the substrate length in the soluble stromal AtAOR, an enzyme that only reduces short-chain α,β unsaturated carbonyls. The AtAOR specificity for small compounds probably results from the presence of a long loop located in the vicinity of the binding site of AtAOR, which could prevent the binding of large molecules.

By comparison with other QORs that are stable dimers, all our results indicate that ceQORH is a monomer in solution and is active as a monomer. Oligomerization observed in crystals and in AUC experiments only occurs at high protein concentrations under conditions that are probably irrelevant of physiological conditions. Comparison of ceQORH structure with other dimeric QORs shows that the ceQORH dimer is different from the stable dimer of QORs due to structural differences in the Rossmann fold. A dimerization mode similar to that of AtAER is prevented in ceQORH by the orientation of the α-helix Pro254-Met267. Analysis of QOR from C. Burnetii (PDB entry: 3TQH) (Franklin et al., 2015) bound to NADPH, which is also a monomeric QOR supports this explanation as an α-helix equivalent to the ceQORH Pro254-Met267 α-helix is also observed. As ceQORH is associated to the inner membrane of chloroplast envelope (Miras et al., 2007), a monomeric state could increase its catalytic efficiency (higher mobility, better dispersion on the membrane surface) and might have been selected during evolution as this would favor the detoxification rate.

The successful crystallization of ceQORH in the presence of ketotriene and ketodiene raised the question of the physiological significance of this property. Kinetic experiments showed that ceQORH could be inhibited by 13-KODE. However, the inhibition was a slow process taking 400 s to reach completion A IC₅₀ of 36 μM for 13-KODE could be determined in the presence of 25 μM substrate (Figures S5). 13-KODE and 13-KOTE are molecules that are produced during the hypersensitive response (HR) of plants attacked by a pathogen (Andersson et al., 2006). This response leads to local destruction of the plant tissues, preventing the spreading of the pathogen. Ketodiene and ketotriene can accumulate to 2.5–5 nmol per g fresh weight several hours after the hypersensitive response is initiated (Andersson et al., 2006). This value corresponds to 2.5–5 μM assuming a homogeneous distribution in the cell. As these compounds could be concentrated locally, they could reached concentration close to ceQORH IC₅₀ and a first hypothesis might be that ceQORH sequesters ketodi(tri)enes quantitatively. However, this does not seem to be the case for the following reason: ceQORH is a low abundant protein at the cellular level. ceQORH represents about 1% of the chloroplast envelope proteins corresponding to about 50,000 copies per chloroplast, i.e., ~1 μM assuming the protein is soluble. This is barely compatible with an efficient sequestering of sub-micromolar concentrations of ketodiene and ketotriene. Another model can be proposed. Some RES oxylipins are signaling molecules (Farmer and Mueller, 2013). Upon increase of the ketodiene and ketotriene levels, following the triggering of the HR (Andersson et al., 2006), ceQORH would become inactivated by these molecules and would then be unable to metabolize γ-ketols. γ-ketols would in turn accumulate and contribute either to signaling or to an additional chemical defense of the plant. ceQORH may thus have a dual function, detoxification of γ-ketols under normal conditions and indirect contribution to chemical defense/signaling after pathogen attack. This would be reminiscent of the “floodgate” hypothesis proposed for H₂O₂ signaling where inhibition of 2-Cys peroxiredoxins occurs at high concentration of H₂O₂ (Wood et al., 2003).

More work is required to determine how ceQORH handles 13-KODE, 13-KOTE and γ-ketols in vivo, and whether ceQORH is involved during the HR response to pathogen attacks.

SMM performed experiments, analyzed data and wrote the article. GC designed and performed experiments, analyzed data, and wrote the article. CG performed experiments. JLF and NR participated to the article writing. DC designed and performed experiments, analyzed data, and wrote the article. All authors read and approved the final manuscript.