Fakhima K. Mukhitova

Unprecedented pathogen‐inducible complex oxylipins from flax – linolipins A and B

Oxylipins constitute a large family of bioregulators, biosynthesized via unsaturated fatty acid oxidation. This study reports the detection of an unprecedented family of complex oxylipins from flax leaves. Two major members of this family, compounds 1 and 2, were isolated and purified. Their structures were evaluated using NMR and MS analyses. Both compounds were identified as monogalactosyldiacylglycerol species. Compound 1 contains one α‐linolenoyl residue and one residue of (9Z,11E,1′Z,3′Z)‐12‐(1′,3′‐hexadienyloxy)‐9,11‐dodecadienoic, (ω5Z)‐etherolenic acid at positions sn‐1 and sn‐2, respectively. Compound 2 possesses (ω5Z)‐etherolenic acid residues at both position sn‐1 and position sn‐2. We suggest the trivial names linolipin A and linolipin B for compounds 1 and 2, respectively, and the collective name linolipins for this new family of complex oxylipins. The linolipin content of flax leaves increased significantly in response to pathogenesis. Thus, accumulation of esterified antimicrobial divinyl ethers may be of relevance to plant defense.

Hydroperoxide lyase cascade in pea seedlings: Non-volatile oxylipins and their age and stress dependent alterations.

The profiles of non-volatile oxylipins of pea (Pisum sativum) seedlings were examined by gas chromatography-mass spectrometry after invitro incubation with α-linolenic acid. The 13-lipoxygenase/hydroperoxide lyase (HPL) products were predominant in the leaves, while the roots possess both 13- and 9-HPL products. Allene oxide synthase (AOS) and divinyl ether synthase (DES) products were not detected in the leaves or in the roots of any age. The HPL cascade produces a diversity of oxylipins, including the compounds (2E)-4-hydroxy-traumatic, (10E)-9,12-dihydroxy-10-dodecenoic and 9,12-dihydroxydodecanoic acids, as well as (2E)-4-hydroxy-2-nonenoic acid, which has not yet been detected in plants. Oxylipin patterns were altered by infection, water deficit, as well as by plant age. Infection caused the specific strong accumulation of azelaic (nonane-1,9-dioic) acid in the leaves. The azelaic acid content in the aged (14 and 18day-old) leaves was significantly higher than in the younger leaves. Water deficit induced the accumulation of (2E)-4-hydroxy-2-nonenoic acid and (2E)-traumatic acid in the roots. Results demonstrate that: (1) the HPL cascade is the predominant branch of the lipoxygenase pathway in pea seedlings; (2) the HPL products may have the regulatory role both in growth control and adaptation.

Oxylipins in the spikemoss Selaginella martensii: Detection of divinyl ethers, 12-oxophytodienoic acid and related cyclopentenones

Green tissues of spikemoss Selaginella martensii Spring possessed the complex oxylipins patterns. Major oxylipins were the products of linoleic and α-linolenic acids metabolism via the sequential action of 13-lipoxygenase and divinyl ether synthase (DES) or allene oxide synthase (AOS). AOS products were represented by 12-oxophytodienoic acid (12-oxo-PDA) isomers. Exceptionally, S. martensii possesses high level of 12-oxo-9(13),15-PDA, which is very uncommon in flowering plants. Separate divinyl ethers were purified after micro-preparative incubations of linoleic or α-linolenic acids with homogenate of S. martensii aerial parts. The NMR data allowed us to identify all geometric isomers of divinyl ethers. Linoleic acid was converted to divinyl ethers etheroleic acid, (11Z)-etheroleic acid and a minority of (ω5Z)-etheroleic acid. With α-linolenate precursor, the specificity of divinyl ether biosynthesis was distinct. Etherolenic and (ω5Z)-etherolenic acids were the prevailing products while (11Z)-etherolenic acid was a minor one. Divinyl ethers are detected first time in non-flowering land plant. These are the first observations of fatty acid metabolism through the lipoxygenase pathway in spikemosses (Lycopodiophyta).

Specificity of oxidation of linoleic acid homologs by plant lipoxygenases

The lipoxygenase-catalyzed oxidation of linoleic acid homologs was studied. While the linoleic acid oxidation by maize 9-lipoxygenase (9-LO) specifically produced (9S)-hydroperoxide, the dioxygenation of (11Z,14Z)-eicosadienoic (20:2) and (13Z,16Z)-docosadienoic (22:2) acids by the same enzyme lacked regio- and stereospecificity. The oxidation of 20:2 and 22:2 by 9-LO afforded low yields of racemic 11-, 12-, 14-, and 15-hydroperoxides or 13- and 17-hydroperoxides, respectively. Soybean 13-lipoxygenase-1 (13-LO) specifically oxidized 20:2, 22:2, and linoleate into (ω6S)-hydroperoxides. Dioxygenation of (9Z,12Z)-hexadecadienoic acid (16:2) by both 9-LO and 13-LO occurred specifically, affording (9S)- and (13S)-hydroperoxides, respectively. The data are consistent with the “pocket theory of lipoxygenase catalysis” (i.e. with the penetration of a substrate into the active center with the methyl end first). Our findings also demonstrate that the distance between carboxyl group and double bonds substantially determines the positioning of substrates within the active site.

Isolation and structure elucidation of linolipins C and D, complex oxylipins from flax leaves

Two complex oxylipins (linolipins C and D) were isolated from the leaves of flax plants inoculated with phytopathogenic bacteria Pectobacterium atrosepticum. Their structures were elucidated based on UV, MS and NMR spectroscopic data. Both oxylipins were identified as digalactosyldiacylglycerol (DGDG) molecular species. Linolipin C contains one residue of divinyl ether (ω5Z)-etherolenic acid and one α-linolenate residue at sn-1 and sn-2 positions, respectively. Linolipin D possesses two (ω5Z)-etherolenic acid residues at both sn-1 and sn-2 positions. The rapid formation (2-30min) of linolipins C and D alongside with linolipins A and B occurred in the flax leaves upon their damage by freezing-thawing.

Stereospecific biosynthesis of (9S,13S)-10-oxo-phytoenoic acid in young maize roots.

Profiling of oxylipins from young maize roots revealed complex patterns of products mainly originating from the combined actions of 9- and 13-lipoxygenases and allene oxide synthase (AOS). A distinctive feature was the high content of the cyclopentenone 10-oxo-11-phytoenoic acid (10-oxo-PEA). Incubations with [1-14C]linoleic acid led to the formation of the α-ketols 13-hydroxy-12-oxo-9-octadecenoic acid and 9-hydroxy-10-oxo-12-octadecenoic acid as well as the cyclopentenones 12-oxo-10-phytoenoic acid (12-oxo-PEA) and 10-oxo-PEA in a ratio of 10:2:1:3. Chiral phase radio-HPLC showed that the labeled 10-oxo-PEA was mainly (93%) due to the 9S,13S-enantiomer, whereas 12-oxo-PEA was racemic. Recombinant maize AOS CYP74A19 (ZmAOS2) converted linoleic acid 9(S)-hydroperoxide (9-HPOD) into an allene oxide, 9,10-epoxy-10,12-octadecadienoic acid (9,10-EOD), which did not undergo cyclization but was solely hydrolyzed into the α-ketol. A cyclase activity promoting the conversion of 9,10-EOD into (9S,13S)-10-oxo-PEA was detected in the 10(5)×g supernatant prepared by differential centrifugation of the maize root homogenate. The data obtained suggested the existence of a new type of allene oxide cyclase, which is active towards an allene oxide formed from a 9-lipoxygenase-derived hydroperoxide.

Sterol binding by methyl‐β‐cyclodextrin and nystatin – comparative analysis of biochemical and physiological consequences for plants

The dependence of membrane function on its sterol component has been intensively studied with model lipids and isolated animal membranes, but to a much lesser extent with plant membranes. Depleting membrane sterols could be predicted to have a strong effect on membrane activity and have harmful physiological consequences. In this study, we characterized membrane lipid composition, membrane permeability for ions, some physiological parameters, such as H2O2 accumulation, formation of autophagosomal vacuoles, and expression of peroxidase and autophagic genes, and cell viability in the roots of wheat (Triticum aestivum L.) seedlings in the presence of two agents that specifically bind to endogenous sterols. The polyene antibiotic nystatin binds to endogenous sterols, forming so‐called ‘nystatin pores’ or ‘channels’ in the membrane, and methyl‐β‐cyclodextrin has the capacity to sequester sterols in its hydrophobic core. Unexpectedly, although application of both methyl‐β‐cyclodextrin and nystatin reduced the sterol content, their effects on membrane permeability, oxidative status and autophagosome formation in roots differed dramatically. For comparison, we also tested the effects of the antibiotic gramicidin S, which does not bind to sterols but forms nonspecific channels in the membrane. Gramicidin S considerably increased membrane permeability, caused oxidative stress, and reduced cell viability. Our results suggest that a decrease in the sterol content is, in itself, not sufficient to have deleterious effects on a cell. The disturbance of membrane integrity, rather than the decrease in the sterol content, is responsible for the toxicity of sterol‐binding compounds.

Screening of divinyl ether synthase activity in nonphotosynthetic tissue of asparagales

116 The current level of knowledge on plant genomics indicates that the lipoxygenase pathway in plants is regulated by a wide variety of genes. Molecular clon ing studies are far behind the pace of genome sequenc ing. Biochemical studies of recombinant enzymes are even more behind. Therefore, it is relevant to study not only catalysis performed by recombinant enzymes but also metabolism in vitro in plants with unstudied or poorly studied genomes. It is known that lipoxygenase metabolic signalling plays a special role in nonphoto synthesizing organs of higher plants and that the met abolic pathways in these organs are highly diverse. CYP74 family enzymes (P450 superfamily) are the key enzymes of the lipoxygenase signaling cascade in plants, which largely determine its orientation. Today, three major categories of these enzymes are known: allene oxide synthases (dehydratases), hydroperoxide lyases (isomerases), and divinyl ether synthases (dehy dratases) [1–3]. All these enzymes regulate the metab olism of fatty acid hydroperoxides, the primary prod ucts of lipoxygenase catalysis. The genes encoding allene oxide synthases and hydroperoxide lyases were found in all sequenced plant genomes. These two enzymes are widely spread in plants. However, the activity of divinyl ether synthase and divinyl ethers were found in phylogenetically distant plant species from brown algae to Solanaceae [4–12]. The lack of DES activity in certain species can be explained by either the absence of the DES gene in this species or by a low level of expression of this gene. The second pos sibility is supported by the data on the pathogen inducible DES expression in some species as well as by the fact that divinyl ethers exhibit antimicrobial activ ity [8, 9]. In the relatively few plant species in which the activity of DES is high, the DES gene expression is constitutive. In particular, DES genes are constitu tively expressed in the bulbs of garlic, leaves of Ranun culaceae species and flax, and roots of Solanaceae spe cies [8, 10, 11]. The DESs studied are not identical, differing in both the substrate specificity and in the geometric isomerism of products. In this paper, we report the finding of a novel divinyl ether synthase in nonphotosynthetic tissues of some plants of the order Asparagales. Previously, we showed that some plants of the order Asparagales, namely, the garlic Allium sati vum L. [12–14] and the lily of the valley Convallária majális L. [15], exhibit divinyl ether synthase activity. In this context, we have continued the screening of divinyl ether synthase activity in the roots and bulbs of the iris (Iris germanica L.), gladiolus (Gladiolus com munis L.), crocus (Crocus vernus L.), and hyacinth (Hyacinthus orientalis L.). The plant material was sup plied by the Zeester Bloemen en Planten BV Company (the Netherlands).

Hexadecanoid pathway in plants: Lipoxygenase dioxygenation of (7Z,10Z,13Z)-hexadecatrienoic acid

Abstract7,10,13-Hexadecatrienoic acid (16:3) is abundant in many plant species. However, its metabolism through the lipoxygenase pathway is not sufficiently understood. The goal of present work was to investigate the oxygenation of 16:3 by different plant lipoxygenases and to study the occurrence of oxygenated derivatives of 16:3 in plant seedlings. The recombinant maize 9-lipoxygenase specifically converted 16:3 into (7S)-hydroperoxide. Identification of this novel oxylipin was substantiated by data of GC-MS, LC-MS/MS, 1H-NMR, and 2D-COSY as well as by deuterium labeling from [2H6]16:3. Soybean lipoxygenase 1 produced 91% (11S)-hydroperoxide and 6% racemic 14-hydroperoxide. Recombinant soybean lipoxygenase 2 (specifically oxidizing linoleate into 13-hydroperoxide) lacked any specificity towards 16:3. Lipoxygenase 2 produced 7-, 8-, 10-, 11-, 13-, and 14-hydroperoxides of 16:3, as well as a significant amount of bis-allylic 9-hydroperoxide. Seedlings of several examined plant species possessed free hydroxy derivatives of 16:3 (HHTs), as well as their ethyl esters. Interestingly, HHTs occur not only in “16:3 plants”, but also in typical “18:3 plants” like pea and soybean seedlings.

Stress-induced changes in membrane sterols in wheat roots

53 Sterols are an important structural element of bioo logical membranes. Unlike animals, higher plants have a complex sterol composition [1], the main comm ponents of which are βsitosterol, stigmasterol, and campesterol. We have previously shown the presence of the main types of phytosterols in wheat roots [2]. Today, the research of the functions of phytosterols has reached a new level. It was established that sterols play not only structural but also regulatory role. It is known that sterols are precursors of plant hormones brassii nosteroids, which regulate plant growth and developp ment [3]. In addition, sterols are directly involved in the formation of sphingolipidd and sterollrich microo domains in membranes. These microdomains can form platforms for the aggregation of signaling comm plexes, including glycosylphosphatidylinositol (GPI)) anchored proteins [4]. It is believed that these strucc tures regulate the transduction of various signals into the cell, including those initiating cell division, differr entiation, and stress responses [5]. Previously, we showed that the binding of sterols by nystatin causes not only a drastic drop in the content of sterols in wheat root cells but also induces changes in the structure of membrane sphingolipids [6]. It can be assumed that stresssinduced changes in the compoo sition of membrane sterols affect the functioning of the membrane and, along with other factors, deterr mine the strategy of the stress response of plant cells. The aim of this study was to analyze the composition and content of sterols in wheat roots under conditions of abiotic stress and to determine the level of express sion of the TaSMT gene, encoding sterol 244CCmethh yltransferase—the key enzyme in the biosynthesis of phytosterols. The stresssinduced changes in the comm position and content of sterols and TaSMT gene activv ity, identified in this study, convincingly demonstrated the involvement of sterols in the stress response of plant cells. Roots of 44dayyold seedlings of the spring wheat (Triticum aestivum L.) cultivar Kazan Jubileinaya were exposed to the following stressor: wounding (cut off the roots of seedlings, 1–6 h), cold (4°C, 1 h), and oxidizing (paraquat treatment, 12 h). The total lipids were extracted from the roots with a mixture of isoproo panol and chloroform [7]. Sterols were analyzed by oneedimensional thinnlayer chromatography (TLC) using the following solvent systems for neutral lipids: (1) hexane–toluene–formic acid (140 : 60 : 1) and (2) hexane : diethyl ether : formic acid (60 : 40 : 1). …