A non-targeted metabolomics analysis identifies wound-induced oxylipins in Physcomitrium patens.

Plant oxylipins are a class of lipid-derived signaling molecules being involved in the regulation of various biotic and abiotic stress responses. A major class of oxylipins are the circular derivatives to which 12-oxo-phytodienoic acid (OPDA) and its metabolite jasmonic acid (JA) belong. While OPDA and its shorter chain homologue dinor-OPDA (dnOPDA) seem to be ubiquitously found in land plants ranging from bryophytes to angiosperms, the occurrence of JA and its derivatives is still under discussion. The bryophyte Physcomitrium patens has received increased scientific interest as a non-vascular plant model organism over the last decade. Therefore, we followed the metabolism upon wounding by metabolite fingerprinting with the aim to identify jasmonates as well as novel oxylipins in P. patens. A non-targeted metabolomics approach was used to reconstruct the metabolic pathways for the synthesis of oxylipins, derived from roughanic, linoleic, α-linolenic, and arachidonic acid in wild type, the oxylipin-deficient mutants of Ppaos1 and Ppaos2, the mutants of Ppdes being deficient in all fatty acids harboring a Δ6-double bond and the C20-fatty acid-deficient mutants of Ppelo. Beside of OPDA, iso-OPDA, dnOPDA, and iso-dnOPDA, three additional C18-compounds and a metabolite being isobaric to JA were identified to accumulate after wounding. These findings can now serve as foundation for future research in determining, which compound(s) will serve as native ligand(s) for the oxylipin-receptor COI1 in P. patens.

Plastidic membrane lipids are oxidized by a lipoxygenase in Lobosphaera incisa.

Green microalgae can accumulate neutral lipids, as part of a general lipid remodeling mechanism under stress such as nitrogen starvation. Lobosphaera incisa is of special interest because of its unique TAG acyl chain composition, especially 20:4 (n-6) can reach up to 21% of dry weight after nitrogen starvation. In order to identify factors that may influence the accumulation of polyunsaturated fatty acids (PUFAs), we identified recently a linoleate 13-lipoxygenase (LiLOX). It shares highest identity with plastidic enzymes from vascular plants and is induced upon nitrogen starvation. Here, we confirmed the localization of LiLOX in the stroma of plastids via transient expression in epithelial onion cells. In order to further characterize this enzyme, we focused on the identification of the endogenous substrate of LiLOX. In this regard, an ex vivo enzymatic assay, coupled with non-targeted analysis via mass spectrometry allowed the identification of MGDG, DGDG and PC as three substrate candidates, later confirmed via in vitro assays. Further investigation revealed that LiLOX has preferences towards the lipid class MGDG, which seems in agreement with its localization in the galactolipid rich plastid. Altogether, this study shows the first characterization of plastidic LOX from green algae, showing preference for MGDGs. However, lipidomics analysis did neither reveal an endogenous LiLOX product nor the final end product of MGDG oxidation. Nevertheless, the latter is a key to understanding the role of this enzyme and since its expression is highest during the degradation of the plastidic membrane, it is tempting to assume its involvement in this process.

Plastidial wax ester biosynthesis as a tool to synthesize shorter and more saturated wax esters.

BACKGROUND: Wax esters (WE) are neutral lipids that consist of a fatty alcohol esterified to a fatty acid. WE are valuable feedstocks in industry for producing lubricants, coatings, and cosmetics. They can be produced chemically from fossil fuel or plant-derived triacylglycerol. As fossil fuel resources are finite, the synthesis of WE in transgenic plants may serve as an alternative source. As chain length and desaturation of the alcohol and acyl moieties determine the physicochemical properties of WE and their field of application, tightly controlled and tailor-made WE synthesis in plants would be a sustainable, beneficial, and valuable commodity. Here, we report the expression of ten combinations of WE producing transgenes in Arabidopsis thaliana. In order to study their suitability for WE production in planta, we analyzed WE amount and composition in the transgenic plants. RESULTS: The transgenes consisted of different combinations of a FATTY ACYL-COA/ACP REDUCTASE (FAR) and two WAX SYNTHASES/ACYL-COA:DIACYLGLYCEROL O-ACYLTRANSFERASES (WSD), namely WSD2 and WSD5 from the bacterium Marinobacter aquaeoleoi. We generated constructs with and without plastidial transit peptides to access distinct alcohol and acyl substrate pools within A. thaliana cells. We observed WE formation with plastid and cytosol-localized FAR and WSD in seeds. A comparative WE analysis revealed the production of shorter and more saturated WE by plastid-localized WE biosynthesis compared to cytosolic WE synthesis. CONCLUSIONS: A shift of WE formation into seed plastids is a suitable approach for tailor-made WE production and can be used to synthesize WE that are mainly derived from mid- and long-chain saturated and monounsaturated substrates.

Sphingolipid long-chain base hydroxylation influences plant growth and callose deposition in Physcomitrium patens.

Sphingolipids are enriched in microdomains in the plant plasma membrane (PM). Hydroxyl groups in the characteristic long-chain base (LCB) moiety might be essential for the interaction between sphingolipids and sterols during microdomain formation. Investigating LCB hydroxylase mutants in Physcomitrium patens might therefore reveal the role of certain plant sphingolipids in the formation of PM subdomains. Physcomitrium patens mutants for the LCB C-4 hydroxylase S4H were generated by homologous recombination. Plants were characterised by analysing their sphingolipid and steryl glycoside (SG) profiles and by investigating different gametophyte stages. s4h mutants lost the hydroxyl group at the C-4 position of their LCB moiety. Loss of this hydroxyl group caused global changes in the moss sphingolipidome and in SG composition. Changes in membrane lipid composition may trigger growth defects by interfering with the localisation of membrane-associated proteins that are crucial for growth processes such as signalling receptors or callose-modifying enzymes. Loss of LCB-C4 hydroxylation substantially changes the P. patens sphingolipidome and reveals a key role for S4H during development of nonvascular plants. Physcomitrium patens is a valuable model for studying the diversification of plant sphingolipids. The simple anatomy of P. patens facilitates visualisation of physiological processes in biological membranes.

Convergence of sphingolipid desaturation across over 500 million years of plant evolution.

For plants, acclimation to low temperatures is fundamental to survival. This process involves the modification of lipids to maintain membrane fluidity. We previously identified a new cold-induced putative desaturase in Physcomitrium (Physcomitrella) patens. Lipid profiles of null mutants of this gene lack sphingolipids containing monounsaturated C24 fatty acids, classifying the new protein as sphingolipid fatty acid denaturase (PpSFD). PpSFD mutants showed a cold-sensitive phenotype as well as higher susceptibility to the oomycete Pythium, assigning functions in stress tolerance for PpSFD. Ectopic expression of PpSFD in the Atads2.1 (acyl coenzyme A desaturase-like 2) Arabidopsis thaliana mutant functionally complemented its cold-sensitive phenotype. While these two enzymes catalyse a similar reaction, their evolutionary origin is clearly different since AtADS2 is a methyl-end desaturase whereas PpSFD is a cytochrome b5 fusion desaturase. Altogether, we suggest that adjustment of membrane fluidity evolved independently in mosses and seed plants, which diverged more than 500 million years ago.

The Fifth WS/DGAT Enzyme of the Bacterium Marinobacter aquaeolei VT8.

Wax esters (WE) belong to the class of neutral lipids. They are formed by an esterification of a fatty alcohol and an activated fatty acid. Dependent on the chain length and desaturation degree of the fatty acid and the fatty alcohol moiety, WE can have diverse physicochemical properties. WE derived from monounsaturated long-chain acyl moieties are of industrial interest due to their very good lubrication properties. Whereas WE were obtained in the past from spermaceti organs of the sperm whale, industrial WE are nowadays mostly produced chemically from fossil fuels. In order to produce WE more sustainably, attempts to produce industrial WE in transgenic plants are steadily increasing. To achieve this, different combinations of WE producing enzymes are expressed in developing Arabidopsis thaliana or Camelina sativa seeds. Here we report the identification and characterization of a fifth wax synthase from the organism Marinobacter aquaeolei VT8, MaWSD5. It belongs to the class of bifunctional wax synthase/acyl-CoA:diacylglycerol O-acyltransferases (WSD). The protein was purified to homogeneity. In vivo and in vitro substrate analyses revealed that MaWSD5 is able to synthesize WE but no triacylglycerols. The protein produces WE from saturated and monounsaturated mid- and long-chain substrates. Arabidopsis thaliana seeds expressing a fatty acid reductase from Marinobacter aquaeolei VT8 and MaWSD5 produce WE. Main WE synthesized are 20:1/18:1 and 20:1/20:1. This makes MaWSD5 a suitable candidate for industrial WE production in planta.

High-level accumulation of oleyl oleate in plant seed oil by abundant supply of oleic acid substrates to efficient wax ester synthesis enzymes.

BACKGROUND: Biotechnology enables the production of high-valued industrial feedstocks from plant seed oil. The plant-derived wax esters with long-chain monounsaturated acyl moieties, like oleyl oleate, have favorite properties for lubrication. For biosynthesis of wax esters using acyl-CoA substrates, expressions of a fatty acyl reductase (FAR) and a wax synthase (WS) in seeds are sufficient. RESULTS: For optimization of the enzymatic activity and subcellular localization of wax ester synthesis enzymes, two fusion proteins were created, which showed wax ester-forming activities in Saccharomyces cerevisiae. To promote the formation of oleyl oleate in seed oil, WSs from Acinetobactor baylyi (AbWSD1) and Marinobacter aquaeolei (MaWS2), as well as the two created fusion proteins were tested in Arabidopsis to evaluate their abilities and substrate preference for wax ester production. The tested seven enzyme combinations resulted in different yields and compositions of wax esters. Expression of a FAR of Marinobacter aquaeolei (MaFAR) with AbWSD1 or MaWS2 led to a high incorporation of C18 substrates in wax esters. The MaFAR/TMMmAWAT2-AbWSD1 combination resulted in the incorporation of more C18:1 alcohol and C18:0 acyl moieties into wax esters compared with MaFAR/AbWSD1. The fusion protein of a WS from Simmondsia chinensis (ScWS) with MaFAR exhibited higher specificity toward C20:1 substrates in preference to C18:1 substrates. Expression of MaFAR/AbWSD1 in the Arabidopsis fad2 fae1 double mutant resulted in the accumulation of oleyl oleate (18:1/18:1) in up to 62 mol% of total wax esters in seed oil, which was much higher than the 15 mol% reached by MaFAR/AbWSD1 in Arabidopsis Col-0 background. In order to increase the level of oleyl oleate in seed oil of Camelina, lines expressing MaFAR/ScWS were crossed with a transgenic high oleate line. The resulting plants accumulated up to >40 mg g seed-1 of wax esters, containing 27-34 mol% oleyl oleate. CONCLUSIONS: The overall yields and the compositions of wax esters can be strongly affected by the availability of acyl-CoA substrates and to a lesser extent, by the characteristics of wax ester synthesis enzymes. For synthesis of oleyl oleate in plant seed oil, appropriate wax ester synthesis enzymes with high catalytic efficiency and desired substrate specificity should be expressed in plant cells; meanwhile, high levels of oleic acid-derived substrates need to be supplied to these enzymes by modifying the fatty acid profile of developing seeds.

Cyanophage-encoded lipid desaturases: oceanic distribution, diversity and function.

Cyanobacteria are among the most abundant photosynthetic organisms in the oceans; viruses infecting cyanobacteria (cyanophages) can alter cyanobacterial populations, and therefore affect the local food web and global biochemical cycles. These phages carry auxiliary metabolic genes (AMGs), which rewire various metabolic pathways in the infected host cell, resulting in increased phage fitness. Coping with stress resulting from photodamage appears to be a central necessity of cyanophages, yet the overall mechanism is poorly understood. Here we report a novel, widespread cyanophage AMG, encoding a fatty acid desaturase (FAD), found in two genotypes with distinct geographical distribution. FADs are capable of modulating the fluidity of the host's membrane, a fundamental stress response in living cells. We show that both viral FAD (vFAD) families are Δ9 lipid desaturases, catalyzing the desaturation at carbon 9 in C16 fatty acid chains. In addition, we present a comprehensive fatty acid profiling for marine cyanobacteria, which suggests a unique desaturation pathway of medium- to long-chain fatty acids no longer than C16, in accordance with the vFAD activity. Our findings suggest that cyanophages are capable of fiddling with the infected host's membranes, possibly leading to increased photoprotection and potentially enhancing viral-encoded photosynthetic proteins, resulting in a new viral metabolic network.

Synthesis of oleyl oleate wax esters in Arabidopsis thaliana and Camelina sativa seed oil.

Seed oil composed of wax esters with long-chain monoenoic acyl moieties represents a high-value commodity for industry. Such plant-derived sperm oil-like liquid wax esters are biodegradable and can have excellent properties for lubrication. In addition, wax ester oil may represent a superior substrate for biodiesel production. In this study, we demonstrate that the low-input oil seed crop Camelina sativa can serve as a biotechnological platform for environmentally benign wax ester production. Two biosynthetic steps catalysed by a fatty alcohol-forming acyl-CoA reductase (FAR) and a wax ester synthase (WS) are sufficient to achieve wax ester accumulation from acyl-CoA substrates. To produce plant-derived sperm oil-like liquid wax esters, the WS from Mus musculus (MmWS) or Simmondsia chinensis (ScWS) were expressed in combination with the FAR from Mus musculus (MmFAR1) or Marinobacter aquaeolei (MaFAR) in seeds of Arabidopsis thaliana and Camelina sativa. The three analysed enzyme combinations Oleo3:mCherry:MmFAR1∆c/Oleo3:EYFP:MmWS, Oleo3:mCherry:MmFAR1∆c/ScWS and MaFAR/ScWS showed differences in the wax ester molecular species profiles and overall biosynthetic performance. By expressing MaFAR/ScWS in Arabidopsis or Camelina up to 59% or 21% of the seed oil TAGs were replaced by wax esters, respectively. This combination also yielded wax ester molecular species with highest content of monounsaturated acyl moieties. Expression of the enzyme combinations in the Arabidopsis fae1 fad2 mutant background high in oleic acid resulted in wax ester accumulation enriched in oleyl oleate (18:1/18:1 > 60%), suggesting that similar values may be obtained with a Camelina high oleic acid line.

Lipoxygenase-derived 9-hydro(pero)xides of linoleoylethanolamide interact with ABA signaling to arrest root development during Arabidopsis seedling establishment.

Ethanolamide-conjugated fatty acid derivatives, also known as N-acylethanolamines (NAEs), occur at low levels (µg per g) in desiccated seeds, and endogenous amounts decline rapidly with seedling growth. Linoleoylethanolamide (NAE18:2) is the most abundant of these NAEs in seeds of almost all plants, including Arabidopsis thaliana. In Arabidopsis, NAE18:2 may be oxidized by lipoxygenase (LOX) or hydrolyzed by fatty acid amide hydrolase (FAAH) during normal seedling establishment, and this contributes to the normal progression of NAE depletion that is coincident with the depletion of abscisic acid (ABA). Here we provide biochemical, genetic and pharmacological evidence that a specific 9-LOX metabolite of NAE18:2 [9-hydro(pero)xy linoleoylethanolamide (9-NAE-H(P)OD)] has a potent negative influence on seedling root elongation, and acts synergistically with ABA to modulate the transition from embryo to seedling growth. Genetic analyses using mutants in ABA synthesis (aba1 and aba2), perception (pyr1, pyl1, pyl2, pyl4, pyl5 and pyl8) or transcriptional activation (abi3-1) indicated that arrest of root growth by 9-NAE-H(P)OD requires an intact ABA signaling pathway, and probably operates to increase ABA synthesis as part of a positive feedback loop to modulate seedling establishment in response to adverse environmental conditions. These results identify a specific, bioactive ethanolamide oxylipin metabolite of NAE18:2, different from those of ethanolamide-conjugated linolenic acid (NAE18:3), as well as a molecular explanation for its inhibitory action, emphasizing the oxidative metabolism of NAEs as an important feature of seedling development.