Caleosin/peroxygenases: multifunctional proteins in plants

Abstract

Background: Caleosin/peroxygenases (CLO/PXGs) are a family of multifunctional proteins that are ubiquitous in land plants and are also found in some fungi and green algae. CLO/PXGs were initially described as a class of plant lipid-associated proteins with some similarities to the oleosins that stabilize lipid droplets (LDs) in storage tissues, such as seeds. However, we now know that CLO/PXGs have more complex structures, distributions and functions than oleosins. Structurally, CLO/PXGs share conserved domains that confer specific biochemical features, and they have diverse localizations and functions.

Scope: This review surveys the structural properties of CLO/PXGs and their biochemical roles. In addition to their highly conserved structures, CLO/PXGs have peroxygenase activities and are involved in several aspects of oxylipin metabolism in plants. The enzymatic activities and the spatiotemporal expression of CLO/PXGs are described and linked with their wider involvement in plant physiology. Plant CLO/PXGs have many roles in both biotic and abiotic stress responses in plants and in their responses to environmental toxins. Finally, some intriguing developments in the biotechnological uses of CLO/PXGs are addressed.

Conclusions: It is now two decades since CLO/PXGs were first recognized as a new class of lipid-associated proteins and only 15 years since their additional enzymatic functions as a new class of peroxygenases were discovered. There are many interesting research questions that remain to be addressed in future physiological studies of plant CLO/PXGs and in their recently discovered roles in the sequestration and, possibly, detoxification of a wide variety of lipidic xenobiotics that can challenge plant welfare.

Keywords: Caleosin; lipid droplets; oxylipins; peroxygenase; plant lipids; stress responses.

Fig. 1.

Presence of caleosin/peroxygenase (CLO/PXG) sequences across the Viridiplantae and their estimated evolutionary divergence times. The major taxa that contain CLO/PXG sequences are shown as green-shaded boxes. Individual species with one or more CLO/PXG sequence are shown in blue, whereas species in which CLO/PXG sequences are definitely absent from their genomes are shown in red. The estimated evolutionary divergence times [in million years ago (My)] of selected key taxa are given. Major taxa with red asterisks are those with good evidence of monophyletic status, whereas taxa without asterisks are probably polyphyletic or paraphyletic. Data are from Rahman et al. (2018b).

Fig. 2.

Phylogenetic analysis of 67 caleosin/peroxygenase (CLO/PXG) sequences from 34 species across the Viridiplantae. The sequences separate into four clearly distinct clusters that are labelled as follows: (A) chlorophyte and streptophyte green algae; (B) non-angiosperm embryophytes; (C) H-isoform angiosperms; and (D) L-isoform angiosperms. Data are from Rahman et al. (2018b). For CLO/PXG gene names, please refer to Table 1.

Fig. 3.

Speculative models of possible lipid associations of plant caleosin/peroxygenases (CLO/PXGs). (A) Possible association of CLO/PXG to bilayer membranes via its transmembrane domain, which includes a proline-knot motif. In the case of a plasmalemma-bound CLO/PXG, the C-terminal domain of CLO/PXG, with its kinase phosphorylation sites and disulfide bonds, is exposed outside the cell, suggesting possible roles in stress-induced signalling. In contrast, the N-terminal domain is inside the cell, with its Ca⁺²-binding domain (residues 75–86) and the essential His70 residue for PXG activity. However, the two essential His residues His70 and His138 are found on opposite sides of the transmembrane domain, as predicted in all the algorithms used, raising questions about the structural elements and crucial residues of the active site of PXG involved in the mechanistic features of this atypical haemoprotein. (B) Association of CLO/PXG with the monolayer membrane of a lipid droplet (LD). In this model, the lipid domain folds back out of the LD, meaning that both the N- and C-termini of CLO/PXG are found outside the LD, and the two essential His residues for PXG activity are found on the same side. Although these are speculative models, they have provided useful structural predictions that can be tested in the laboratory.

Fig. 4.

Putative catalytic mechanism of a plant caleosin/peroxygenase (CLO/PXG). Hydroperoxide fatty acids (e.g. 13-HPOT) are generated from linolenic acid by 13-lipoxygenases. In the first step, the iron atom attacks the O–O bond of the hydroperoxide group and fixes one oxygen atom by heterolytic cleavage, leading to the formation of a reversible oxo-haem complex with an iron(II) oxide (Fe–O) polar bond in the active site of the peroxygenase (PXG). This results in the reduction of the fatty acid hydroperoxide (13-HPOT) to its corresponding hydroxide (13-HOT). This reaction constitutes the fatty acid hydroperoxide-reductase activity of PXG. In the second step, the PXG transfers an oxygen atom via two competing pathways, A and B. In pathway A, the PXG possibly transfers the O- to a C=C double bond in the same unsaturated fatty acid hydroxide (13-HOT) molecule, resulting from the first step, by a mechanism known as intramolecular transfer, leading to the formation of its corresponding hydroxyl-epoxy fatty acid, e.g. 13-hydroxy-15,16-epoxy OD. In pathway B, the PXG transfers the O- to another oxidizable substrate by an intramolecular transfer, thereby catalysing the co-oxidation reactions, e.g. the sulfoxidation of sulfide-containing molecules and the epoxidation of unsaturated fatty acids. These activities generate a characteristic signature of mono-, di- and poly-epoxy fatty acids. This scheme is adapted from Blée et al. (1993).