A. G. Vereshchagin

Lipid fatty acid composition of potato plants transformed with the Δ12-desaturase gene from cyanobacterium

Potato (Solanum tuberosum L.) plants were transformed with the desA gene encoding Δ12 acyl-lipid desaturase in the cyanobacterium Synechocystis sp. PCC 6803. To evaluate the efficiency of this gene expression in the plant, its sequence was translationally fused with the sequence of the reporter gene encoding thermostable lichenase. A comparison of native and hybrid gene expression showed that lichenase retained its activity and thermostability within the hybrid protein, whereas desaturase retained its capability of inserting the double bond in fatty acid (FA) chains and, thus, to modify their composition in membrane lipids. In most transformed plants, shoots contained higher amounts of polyunsaturated FAs, linoleic and linolenic (by 39–73 and 12–41%, respectively). The total absolute content of unsaturated FAs was also higher in transformants by 20–42% as compared to wild-type plants. When transformed plants were severely cooled (to −7°C), the rate of their membrane lipid peroxidation was not enhanced, whereas in wild-type plants, it increased substantially (by 25%) under such conditions. These results could indicate a higher tolerance of transformed plants to low temperatures and the oxidative stress induced by hypothermia.

Determination of the Positional-Species Composition of Plant Reserve Triacylglycerols by Partial Chemical Deacylation

To determine the positional-species composition (PSC) of the sunflower oil triacylglycerols (TAGs), these TAGs were separated in a native state by preparative TLC on alumina and subjected to a partial chemical deacylation with the Grignard reagent under anhydrous conditions. The rac-1,2-diacylglycerols (DAGs) formed in this reaction were identified by comparing their TLC mobilities with the respective standard separated from the products of enzymatic hydrolysis of lecithin. Subsequently, the DAGs as their borate complexes were isolated from the reaction mixture by preparative TLC on silica gel. The quantitative fatty acid composition of both the DAGs and the initial TAGs was determined by capillary GLC. The PSC of TAGs calculated from the data obtained using a computer program was consistent with the data on sunflower TAG composition reported by other researchers using an independent method. Thus, the technique developed here makes it possible to obtain reliable data on the concentration of all TAG positional species in the plant tissue.

Occurrence of fatty acid lower-alkyl esters in euonymus fruits

Small amounts of a mixture of fatty acid lower-alkyl esters (FALAEs) were obtained from chloroform extracts of the fruit arils and seeds of four euonymus species (Euonymus sp., Celastraceae). The FALAEs were the products of biosyntheses in the cell rather than experimental artifacts. By using GC-MS, this mixture was shown to contain a total of 19 individual FALAE species comprising four separate fractions, viz. methyl (FAMEs), ethyl (FAEEs), n-propyl, and n-butyl FA esters. Fruit FALAEs included mainly FAEEs and, to a lesser extent, FAMEs, while the n-propyl and n-butyl FA esters, which occurred less frequently, were found here for the first time as the plant products. The FALAE acid components included C14-C18 saturated, mono-, di-, and trienoic FAs with the predominance of ubiquitous linoleic, oleic, palmitic, and, in some cases, also α-linolenic acid. The indices of qualitative and quantitative composition of separate FALAE fractions varied considerably depending on the plant species, fruit part (aril or seed), and the extent of fruit maturity. It can be supposed that, in some euonymus species, FAMEs and FAEEs are formed at the expense of the same FA pool characteristic for a given species. As a whole, euonymus FALAEs and triacylglycerols seem to be synthesized from different FA pools. Discussed is the physiological significance of FALAE biosynthesis in plant metabolism, possible pathways of this biosynthesis, as well as the perspectives of further investigations of FALAEs of plant origin.

Occurrence of two different systems of triacylglycerol formation in sea buckthorn fruit mesocarp

Summary Separate geographic forms of sea buckthorn ( Hippophae rhamnoides L.) are characterized not only by different composition of triacylglycerols (TAGs) of their fruit mesocarp oil, but also by a different biosynthetic pattern of the TAGs. Siberian, Central Asian, and Baltic forms contain TAGs, which include mostly hexadecenoic (C 16:1 ) and palmitic (C 16:0 ) fatty acid (FA) residues and are synthesized according to the theory of 1,3-Random, 2-Random Distribution of FAs between the respective OH groups of glycerol. At the same time, the mechanism of formation of TAGs of the Caucasian form rich in the residues of C 16:0 and octadecenoic acid (C 18:1 ) is in accordance with the theory of Restricted Random Distribution of FAs. The occurrence of two different patterns of TAG biosynthesis in the mesocarp of the same biological object is demonstrated here for the first time. As for seed oils, the geographic forms studied here virtually do not differ from each other in the FA composition of their TAGs.

Distribution of unusual fatty acids in the triacylglycerols of sea buckthorn mesocarp oil

The structure of unusual fatty acid (FA) components of triacylglycerols (TAGs) of mature sea buckthorn (Hippophae rhamnoides L.) mesocarp oil was determined by GLC and MS, and the positional-species composition (PSC) of these TAGs was estimated using the methods of TAG chemical deacylation, TLC, GLC, and computer calculation. It was shown that the unusual FAs comprised n-cis-Δ9-hexadecenoic, n-cis-Δ9,12-hexadecadienoic (palmitolinoleic), and n-cis-Δ11-octadecenoic (cis-vaccenic) acids. The hexadecenoic acid predominated in the oil, and in its distribution in TAGs, it was similar to the total FAs differing from them only in some prevalence in the triunsaturated TAGs and in the TAGs with a shorter acyl chain, as well as in the sn-2 position of TAGs. Palmitolinoleic (16:2) acid comprised only 5% of total FAs, and it was exclusively concentrated in the sn-2 position of TAGs. As regards its distribution between various positional types and forms of TAGs, the 16:2 acid was similar to oleate and total FAs. As compared to the total TAGs, the TAGs with 16:2 acid were characterized by a lower FA chain length as well as by a highest unsaturation. The TAGs with vaccenic acid (V-TAGs) considerably exceeded O-TAGs, i.e., the TAGs containing oleic acid, another 18:1 positional isomer, both in their content in the total TAGs and in their unsaturation. In the composition of positional types and fractions of various unsaturation, O-TAGs were similar to the total TAGs, while V-TAGs were characterized by a very unusual structure, viz., a very high triunsaturated TAG level and an extremely low concentration of 1,3-disaturated-2-monounsaturated TAGs. In addition, oleic acid, like most other unsaturated FAs, was incorporated predominantly in the sn-2 position of TAGs, while vaccenic acid, being also unsaturated, was nevertheless by 90% concentrated in the sn-1,3 positions of V-TAGs. Unusual FAs were related to each other in the mechanism of their biosynthesis. In fact, hexadecenoic acid biosynthesis produced by palmitic acid desaturation, was, on the one hand, further desaturated forming palmitolinoleic acid, and, on the other hand, converted to vaccenic acid via C2 elongation.

Developmental Changes in the Triacylglycerol Composition of Sea Buckthorn Fruit Mesocarp

Summary The aim of this study was to determine the dynamics of the triacylglycerol (TAG) content and the kinetics of TAG formation in maturing sea buckthorn ( Hippophae rhamnoides ) fruit mesocarp as well as to outline a putative route of TAG biosynthesis. Among total TAGS, which included the residues of palmitic (P), hexadecenoic (H), octadecenoic (O), and linoleic (L) fatty acids (FAs), Group I TAGS (PHH + PHP + HHH + PHO +HHO) were formed at a higher rate than those of Groups II (POH + POP + HOH + POO + HOO) and III (PLH + PLP + HLH + PLO + HLO), and the latter accumulated more rapidly than those of Group II. This pattern of kinetics was caused by differences both in the amount of certain FA species available for TAG formation at a given growth step and in the regioselectivity of FAs for esterifying various positions of a glycerol derivative. Experimental data on TAG composition (confirmed by other authors) were close to those calculated according to the theory of 1,3-Random, 2-Random Distribution of FAs between the respective OH moieties of glycerol. A presumed novel pathway of Group I, II, and III TAG biosynthesis based on these data and consistent with the above theory involves the formation of a pool of five 1,3-diacyl TAG precursors and their ensuing acylation with H, O, and L, respectively.

Preparation of plates with a permanent adsorbent layer and their application in the analytical thin-layer chromatography of lipids.

Experimental conditions for preparing the thin-layer chromatographic plates with a permanent adsorbent layer (PAL) have been established; the particles of this layer are firmly bound to each other and to the glass support by means of fused glass powder. To prepare the PAL plates, a mixture of Woelm silica gel and glass powder (1:3, w/w) of particle size 7+/-1 mum was suspended in toluene and spread on the plates, yielding PALs of thickens 150-250 mum. The plates were heated in an electric furnace at 675 degrees for 20 min. In the separation of neutral lipids, a PAL thus prepared is equal in efficiency and selectivity to silica gel layers prepared in the usual manner.

Increased content of very-long-chain fatty acids in the lipids of halophyte vegetative organs.

Qualitative and quantitative compositions of esterified fatty acids (FAs) in the total lipids from the leaves, shoots, and roots of halophile plants, such as suaeda (Suaeda altissima), samphire (Salicornia europaea), and wormwood (Artemisia lerchiana), collected in their natural environments were estimated by GLC techniques. It was shown that the vegetative organs of these halophytes contained 24 FA species, and 16 of them were tentatively identified as the very-long-chain FAs (VLCFAs). There were four VLCFA groups, viz. C20, C21, C22, and C23, each including saturated, mono-, and diunsaturated components; C24 and C25 FAs were also present. The concentration of VLCFAs in the total FAs comprised 4–64%. In vegetative organs of higher plants not subjected to genetic transformation, such a high VLCFA content was found for the first time. Saturated and even-numbered components predominated among the VLCFAs, and the roots exceeded severalfold the above-ground organs in the total VLCFA content. Possible pathways of VLCFA biosynthesis in plants, VLCFA content in the vegetative tissues, and the physiological role of membrane lipid FA composition in the plant salt metabolism are discussed.

Accumulation of neutral acylglycerols during the formation of morphologo-anatomical structure of euonymus Fruits

At three stages of fruit ripening of three euonymus species (Euonymus sp.), growth parameters, the development of morphologo-anatomical structure, and the accumulation of neutral acylglycerols (NAG) of fatty acids were studied. It was established that, in all species studied, in the cells of endosperm and cotyledons, as well as in arillus cells, small oleosomes of similar size were formed, whereas in Euonymus europaeus and E. maximowiczianus large oleosomes differing in their sizes also appeared. Independently of fruit age, dry weight of seeds exceeded that of arils by several times. At the determination of separate NAG classes: TAG (1,2,3-triacyl-sn-glycerols) and acDAG (3-acetyl-1,2-diacyl-sn-glycerols), it was shown that the absolute content of acDAG in seeds was 1.5 orders of magnitude higher than that of TAG; in contrast, in arils TAG were much more abundant than acDAG. Euonymus species differed markedly in the growth pattern and NAG accumulation in seeds and arils.

Fatty acid composition of total lipids from the needles of pine seedlings as related to boron availability

The effect of boron availability on the content of fatty acids (FA) in the total lipids of needles was investigated in two-year-old seedlings of Scots pine (Pinus sylvestris L.). The study was conducted in the forest nursery on the soil deficient in boron. Various rates of boric acid (0.01, 0.03, 0.1, 0.3, 1, 3, and 10 g/m2) were applied to soil three times throughout the growth period. When pine seedlings were supplied with extra boron, the lipid content of the seedling needles declined. The optimal boron supply elevated the content of saturated FA in total needle lipids mostly at the expense of palmitic acid, with a corresponding decline in the level of unsaturated FA and unsaturation index of FA owing to trienoic FA (mainly linolenic and hexadecatrienoic acids). At the same time, the level of monoenoic and to a lesser degree dienoic acids increased. When boron supply of the seedlings was not optimal, the content of unsaturated and low-molecular FA increased.