Specificity of lipoxygenase pathways supports species delineation in the marine diatom genus Pseudo-nitzschia

Abstract

Oxylipins are low-molecular weight secondary metabolites derived from the incorporation of oxygen into the carbon chains of polyunsaturated fatty acids (PUFAs). Oxylipins are produced in many prokaryotic and eukaryotic lineages where they are involved in a broad spectrum of actions spanning from stress and defense responses, regulation of growth and development, signaling, and innate immunity. We explored the diversity in oxylipin patterns in the marine planktonic diatom Pseudo-nitzschia. This genus includes several species only distinguishable with the aid of molecular markers. Oxylipin profiles of cultured strains were obtained by reverse phase column on a liquid chromatograph equipped with UV photodiode detector and q-ToF mass spectrometer. Lipoxygenase compounds were mapped on phylogenies of the genus Pseudo-nitzschia inferred from the nuclear encoded hyper-variable region of the LSU rDNA and the plastid encoded rbcL. Results showed that the genus Pseudo-nitzschia exhibits a rich and varied lipoxygenase metabolism of eicosapentaenoic acid (EPA), with a high level of specificity for oxylipin markers that generally corroborated the genotypic delineation, even among genetically closely related cryptic species. These results suggest that oxylipin profiles constitute additional identification tools for Pseudo-nitzschia species providing a functional support to species delineation obtained with molecular markers and morphological traits. The exploration of the diversity, patterns and plasticity of oxylipin production across diatom species and genera will also provide insights on the ecological functions of these secondary metabolites and on the selective pressures driving their diversification.

Figure 1. Molecular phylogeny of Pseudo-nitzschia species.

Maximum Likelihood tree constructed with rbcL (A) and LSU (B) sequences. The strains in bold have been analysed for oxylipin production. The position specificity of the LOX activity detected for the different strains and the still unidentified compounds are mapped with different colours on the trees.

Figure 2. Major EPA-derived oxylipins characterized in Pseudo-nitzschia species during this study.

Compounds have been identified on the basis of comparison of primary analytical indicators (retention time, UV spectrum, molecular weight and mass/mass fragmentation) with those experimentally determined or calculated in agreement with [21].

Figure 3. Lipoxygenase positional specificity in the genus Pseudo-nitzschia.

Identification is inferred on MS/MS fragmentation of hydroxy-epoxy eicosatetraenoic acids (HEpETEs) derived from LOX-mediated metabolism of EPA. (a) EPA and position of LOX oxidation. (b) Biochemical mechanism leading to specific transformation of primary LOX product (hydroperoxy-eicosapentaenoic acid, HpEPE) to HEpETE. For simplicity, the polyunsaturated chain of EPA is represented by the 1,3-pentadiene moiety that undergoes to enzymatic oxidation. R₁ and R₂ are variable alkyl residues to complement the structure of EPA. (c) Diagnostic ions of different HEpETEs generated by MS/MS fragmentation.

Figure 4. Heat map representation of oxylipin signals in the Pseudo-nitzschia strains.

A semi-quantitative estimate of the individual oxylipins (rows) recorded in the analyzed strains (columns). Color scale represents the ratio between peak areas of individual oxylipins and internal standard (see Materials and Methods). Suffix ‘/r’ followed by number indicates biological replicates of the same strain. Chemical abbreviations are in agreement with Figure 2.