Identification and Functional Characterization of the Moss Physcomitrella patens Δ5-Desaturase Gene Involved in Arachidonic and Eicosapentaenoic Acid Biosynthesis

Abstract

The moss Physcomitrella patens contains high levels of arachidonic acid and lesser amounts of eicosapentaenoic acid. Here we report the identification and characterization of a ⌬ 5 -desaturase from P. patens that is associated with the synthesis of these fatty acids. A full-length cDNA for this desaturase was identified by data base searches based on homology to sequences of known ⌬ 5 -desaturase cDNAs from fungal and algal species. The resulting P. patens cDNA encodes a 480-amino acid polypeptide that contains a predicted N-terminal cytochrome b 5 -like domain as well as three histidine-rich domains. Expression of the enzyme in Saccharomyces cerevisiae resulted in the production of the ⌬ 5 -containing fatty acid arachidonic acid in cells that were provided di-homo-␥-linolenic acid. In addition, the expressed enzyme generated ⌬ 5 -desaturation products with the C20 substrates -6 eicosadienoic and -3 eicosatrienoic acids, but no products were detected with the C18 fatty acid linoleic and ␣-linolenic acids or with the C22 fatty acid adrenic and docosapentaenoic acids. When the corresponding P. patens genomic sequence was disrupted by replacement through homologous recombination, a dramatic alteration in the fatty acid composition was observed, i.e. an increase in di-homo-␥-linolenic and eicosatetraenoic acids accompanied by a concomitant disappearance of the ⌬ 5 -fatty acid arachidonic and eicosapentaenoic acids. In addition, overexpression of the P. patens cDNA in protoplasts isolated from a disrupted line resulted in the restoration of arachidonic acid synthesis. Polyunsaturated fatty acids (PUFAs),4 fatty acids containing 18 or more carbon atoms with two or more methylene-interrupted double bonds in the cis-position, have received increasing attention in recent years because they are considered to have beneficial effects on human health and development when included in the diet (1, 2). Long-chain PUFAs (ՆC20) are not commonly found in angiosperms; however, they are present in some gymnosperms (3). High proportions of PUFA accumulation are found in many algae, mosses, and ferns (4 -6). The function of these long-chain PUFAs in the membranes of lower plants is still unclear, whereas in humans they play a role in eicosanoid metabolism (7). ) and EPA (20:5 ⌬ 5,8,11,14,17 ) are precursors of the short-lived regulatory molecules, the eicosanoids, that comprise the prostaglandins, the leukotrienes, and the thromboxanes (9 -11). These compounds act locally through autocrine or paracrine process on G-protein-linked cell surface receptors. This leads to the activation of various signaling mechanisms that have effects on numerous cellular functions including chemotaxis, vascular permeability, inflammation, vasoconstriction, regulation of the immune system, blood clotting, neurotransmission, and cholesterol metabolism (12)(13)(14). ARA (20:4 ⌬ 5,8,11,14 ) and EPA (20:5 ⌬ 5,8,11,14,17 ) are converted from linoleic acid (18:2 ⌬ 9,12) and ␣-linolenic acid (18:3 ⌬ 9,12,15 ), respectively, by the activities of ⌬ 6-desatura...

The Plackett–Burman Design for Evaluating the Production of Polyunsaturated Fatty Acids by Physcomitrella patens

“…Fatty acid methyl esters (FAMEs) were then extracted in 1 ml of heptane, the organic layer evaporated to dryness by nitrogen gas, and the residue dissolved with 50 ll of heptane before gas chromatography (GC) [11]. GC analysis of FAMEs was conducted using an HP 6890 Series gas chromatograph equipped with 0.25 mm 9 30 m 9 0.25 lM HP-INNOWax capillary column and a flame ionization detector with helium as the carrier gas.…”

“…This knowledge has led to the search for alternate sources of PUFAs in microalgae, lower fungi and other microorganisms [9]. The moss Physcomitrella patens (Funariales, Bryophyta), which is a lower plant, is also capable of producing several PUFAs including LA, GLA, ALA, eicosadienoic acid (EDA), di-homo-c-linolenic acid (DHGLA), ARA and EPA [10,11]. The ability to synthesize fatty acids up to C 20 by bryophytes makes such lower plants a better choice for investigating them as a versatile source of PUFAs.…”

Membrane lipid composition affects plant heat sensing and modulates Ca2+-dependent heat shock response

“…These long-chain polyunsaturated FAs are abundant in P. patens and other algae, compared to higher plants. 23 Their exact role during plant acclimation to higher temperatures is not known. The observation that C16, C18 and C20 FAs displayed distinct patterns, suggests that individual classes of FA have specific roles in maintaining optimal membrane functions.…”

Can mosses serve as model organisms for forest research?

“…Compared to higher plants, P. patens and other mosses possess high amounts of long PUFAs, whose profile is depending on the developmental stage of the plant (Beike et al 2014). This difference is based on the existence of several desaturases as well as elongases in moss which were lost in angiosperm evolution and catalyze alternative pathways in the biosynthesis of these fatty acids (Eiamsa-ard et al 2013;Girke et al 1998;Kaewsuwan et al 2006;Zank et al 2002).…”