CYP74 enzymes differ from other P450 enzymes, in that they have an atypical reaction mechanism that requires neither oxygen nor an NADPH-reductase, and as a consequence have extraordinarily high turnover numbers. Hydroperoxide lyase (HPL) is a member of this CYP74 sub-family and has an important role either in oxylipin metabolism and plant defence. The enzyme cleaves hydroperoxides, formed from the oxygenation of polyunsaturated fatty acids by the action of lipoxygenase (LOX), into an array of volatile and non-volatile products that have direct and indirect defence properties. HPL has similar substrate specificity to other classes of CYP74 enzymes, allene oxide synthase (AOS) and divinyl ether synthase (DES). Unlike HPL, which cleaves hydroperoxides, AOS transforms them into allene oxides, which are finally converted into jasmonic acid; whereas DES converts them into divinyl ethers, showing antifungal properties (Figure 1).

Figure 1: Schematic representation of plant hydroperoxide lyase pathway, responsible for the biosynthesis of C6 and C9 volatile aldehydes and ϖ-oxoacids. Abbreviations: Lox: Lipoxygenase; AOS: Allene Oxide Synthase; AOC: Allene Oxide Cyclase; OPR3: Oxyphytodienoate Reductase; HPL: Hydroperoxide Lyase; JA: Jasmonic Acid; ADH: Alcohol Dehydrogenase; CHAT: Choline Acetyltransferase.

Hydroperoxide lyases (HPL) catalyse the cleavage of hydroperoxides into short chain aldehydes and ϖ-oxo fatty acids (Figure 1). Some HPLs show a strict specificity for 13-hydroperoxides with consequent production of (Z)-3-hexenal and 12-oxo-(Z)-9-dodecenoic acid starting from 13-hydroperoxy-derivatives of linolenic acids [1,2].

Aldehydes produced by HPL can be further converted to the corresponding alcohols by alcohol dehydrogenase, acetylated by acetyltransferase or isomerised with the consequent production of other volatile compounds i.e. (Z)-3-hexenol, (E)-2-hexenal and (Z)-3-hexenyl acetate. All these volatile compounds are important constituents of the green leaf volatiles (GLV) and are rapidly released by plant in response to mechanical damage or herbivores attack [1,2].

Together with 13-HPL, 9/13-HPLs have been reported. They were initially thought to be restricted to the Cucurbitaceae family, even though their occurrence in other plant species such as Medicago spp., rice, almond and grape have been later reported [3-7]. 9/13-HPL is able to use both 9- and 13-hydroperoxides. Starting from 9-hydroperoxyderivatives of linolenic acids, (E,Z)-2,6-nonadienal and C9-oxo-acids are synthesised. Differently from 13-HPL, whose expression is restricted to green tissues, 9/13 are also expressed in underground tissues as in the case of rice HPL (OsHPL1) and the Medicago truncatula 9/13-HPL (MtHPLF) [4,6].

The phylogenetic analysis carried out on a number of plant HPLs (Figure 2) clearly confirms the presence of these two distinct groups inside plant HPLs.

Figure 2: Phylogenetic relatedness among different 9/13- and 13-HPL.
The phylogenetic tree was obtained using the PHILODENDRONPHYLOGENETIC TREE PRINTER program, comparing amino acid sequences of plant HPsL. HPL sequences were from Medicago sativa (MsHPL1, CAB54849;MsHPL2, CAB54848; MsHPL3, CAB54847), Arabidopsis lyrata subsp. lyrata (AlHPL, EFH46497), Zea mays (ZmHPL, AAS47027), Medicago truncatula (MtHPL1, CAC86898.1; MtHPL2, CAC86899; MtHPL3, AAY30368), Lotus japonicum (LjHPL, BAJ78218), Solanum tuberosum (StHPL, ACT64589),Arabidopsis thaliana (AtHPL, AAC69871), Solanum pennellii ( HPL, ABI93819),
Vitis vinifera (VvHPL1, ADP88810; VvHPL2, ADP88811), Citrus jambhiri (CjHPL, BAC55161), Populus trichocarpa (PtHPL, EEE99451), Nicotina tabacum (NtHPL, AAZ39884), Nicotina attenuata (NaHPL, CAC91565), Olea europea (OeHPL, ACD43482), Solanum lycopersicum (SlHPL, NP_001234420), Capsicum annuum (CaHPL, AAK27266), Oryza sativa (OsHPL, AFU07591), Prunus dulcis (PdHPL, CAE18065), Cucumis sativus (CsHPL, AAF64041) and Cucumis melo (CmHPL, AAK54282).

Since CYP74 enzymes compete for the same substrates, their endocellular localisation is an important control point in the biosynthesis of oxylipins.

A plastidial localisation was clearly demonstrated for HPLs and AOSs showing a strict specificity towards 13-hydroperoxides. As far as the members of CYP74C and D subfamilies, which do not show strict substrate specificity, their localisation is not currently completely elucidated. Some 9/13 HPLs, tagged with the fluorescent marker GFP, were visualised by confocal analysis (Figure 3), after a transient expression in tobacco protoplasts. When compared with that of M. truncatula 13-HPL which shows a clear chloroplast localisation, a different localisation for 9/13-HPLs appears evident. Indeed, 9/13- HPLs showed a prevalent microsomal or cytosolic distribution [5,6]. Notably, these enzymes appeared also to be specifically associated with punctuate spots (indicated by arrows, in figure 3) corresponding to lipid droplets. Biochemical analyses reported in previous works [5,6] confirmed confocal microscopy analyses. We hypothesised that such association with lipid droplets could result in the protein conformational changes required for full activation of these enzymes [1,6].

Figure 3: Fluorescence patterns of some GFP-tagged 9/13- and 13- HPLs in tobacco protoplasts.
Nicotiana tabacum cv. SR1 protoplasts, prepared according to Mita et al. [5], were transformed with the following chimeric constructs and imaged by fluorescence microscopy using a LSM Pascal Zeiss confocal laser-microscope. The localisation pattern of three 9/13-HPLs isolated from almond (Prunus dulcis, PdHPL-GFP; a), Medicago truncatula (MtHPLF1-GFP; b), and cucumber (Cucumis sativus, CsHPL-GFP; c) was compared with that of a 13-HPL isolated from Medicago truncatula (MtHPLE1-GFP; d). GFP was detected with the filter set for FITC (505–530 nm) whereas chlorophyll epifluorescence was detected with the filter set for TRITC (>650 nm). The scale bar corresponds to 20 μm.

The products of HPL catalysis are important constituents of green leaf volatiles (GLV). Their levels are very low in intact plants but increase rapidly after wounding or insect feeding. GLV function as stress signal in the same and in neighbouring plants [8-12], triggering the expression of defence genes [13-15]. Recently, Zhang et al. showed that exposing A. thaliana plants to Lima bean volatiles induced considerable transcriptomic changes [12]. Results indicated that either the ethylene or jasmonate signaling pathways were up-regulated (the latter with a weaker response) whereas salicylic acid pathway did not.

In comparison to pathogens, insects are highly complex organisms showing complex and different behaviours. Plants have evolved specific mechanisms to sense insect pressure, walking and eating. A number of reports showed that mechanostimulation of plant tissues trigger the expression of defence genes of the jasmonate pathway. A second layer of perception occurs when insects disrupt plant tissues integrity. Wounding is per se another important signal able to elicit defence responses, leading to the so called wound-induced resistance. Finally, plants are also able to detect herbivores more specifically by the recognition of specific compounds released during feeding. Indeed, a number of studies have shown that insect oral secretions are able to potentiate wound induced response [16,17]. Therefore, the emerging scenario indicates that wounding, direct feeding or volatiles released by injured plants elicit different stress signals. A recent report showed that either generalist or specialist caterpillars were able to greatly reduce the levels of two main HPL products, i.e. (Z)-3-hexenol and (Z)-3-hexenyl acetate, but increased the levels of jasmonate in A. thaliana plants [18]. By converse, either mechanical wounding or aphids feeding elicited the same compounds. Notably, the expression levels of jasmonate biosynthesis genes were up-regulated by insect feeding which resulted in a burst of jasmonate [18]. The authors hypothesised that these changes in signaling dynamics might be due to insects induced transcriptional modifications of critical genes in the HPL branch.

Together with GLV, which can be considered a blend of different volatile compounds, single volatile compounds, i.e. (Z)-3-hexenyl acetate or nonanal, can also elicit defence responses in neighbouring plants [19-21].

GLV primed plants are able to respond more rapidly or more effectively to a subsequent attack [15,22,23]. With this strategy, plants avoid extensive reprogramming and biochemical investments, which can affect both plant physiology and agronomic performances. A recent report showed that the white-backed planthopper (Sogatella furcifera) but not the brown planthopper (Nilaparvata lugens) was able to induce resistance to bacterial blight in rice [24]. In this case, resistance was mediated by a specific HPL isoform encoded by OsHPL2. Over-expression of OsHPL2 in rice plants resulted in higher resistance to bacterial blight. Furthermore, rice plant exposed to (E)-2- hexenal were more resistant to the same pathogen [24]. A direct antifungal and insecticide effects of volatile aldehydes was also reported in previous works [5,25].

Furthermore, signalling mediated by GLV is faster than vascular system and reach other parts of the plant lacking vascular connections with the damaged tissue or located close to it but on different branches [22,23].

Finally, GLV have been shown to have an important role in tritrophic interactions. HPL over-expressing Arabidopsis plants, showing increased levels of GLV, were more attractive to the parasitic wasp Cotesia glomerata, leading to higher mortality of herbivores, as in the case of larvae of the cabbage white butterfly, Pieris rapae. The same plants showed a higher resistance to the gray mold Bortrytis cinerea. On the contrary, antisense suppression of HPL, resulted in lower levels of GLV, lower attraction of parasitoid and higher susceptibility to pathogen [26]. Wei et al. demonstrated that plants use (Z)-3- hexenol, a universally induced GLV, to attract Opius dissitus wasps. This compound helps wasps to locate their preys, as the pea leafminer Liriomyza huidobrensis [27].

Plants utilise hormones and hormone-like compounds as signals to communicate their stress status from local damaged tissues to distal organs within the same plant and to other neighbouring plants. Some of these signalling molecules, as jasmonates, can be systemically translocated, others, as GLV or methyl jasmonate function as airborne signals. The jasmonate signaling cascade is considered a key regulator of plant defence response to arthropods, herbivores and pathogens. However, it’s becoming evident the existence of a complex tuning among different signaling cascades interacting each others on the basis of specific stress signals released by plant and host. The integration of signals from different signaling pathway has the important role to integrate the initial perception (insect walking/feeding) with later events (release of specific insect elicitors) and translate them into an appropriate defence response.

The use of different, tightly controlled signals and different diffusion systems permit the rapid transmission of the stress signal to all the tissues and the reduction of inappropriate expression of costly systemic defences. Recent reports showed the ecological significance of GLV emitted by injured plants on intact neighbouring plants and the impact of primed defence on their fitness and agronomic performances.