ISSN 10214437, Russian Journal of Plant Physiology, 2010, Vol. 57, No. 5, pp. 739–743. © Pleiades Publishing, Ltd., 2010. Original Russian Text © S.P. Makarenko, Yu.M. Konstantinov, V.N. Shmakov, T.A. Konenkina, 2010, published in Fiziologiya Rastenii, 2010, Vol. 57, No.5, pp. 791–795.
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Fatty Acid Composition of Lipids in the Calluses of Two Pine Species Pinus sibirica and Pinus sylvestris S. P. Makarenko, Yu. M. Konstantinov, V. N. Shmakov, and T. A. Konenkina Siberian Institute of Plant Physiology and Biochemistry, Siberian Division, Russian Academy of Sciences, ul. Lermontova 132, Irkutsk, 664033 Russia; fax: 7 (395) 2510754; email:
[email protected] Received May 21, 2010
Abstract—The fatty acid (FA) composition of callus lipids in two pine species, Pinus sibirica Du Tour and P. sylvestris L. was studied. Callus lipids were characterized by a high content of unsaturated FAs: 81.7% in P. sibirica and 63.2% in P. sylvestris. Among them, oleic and linoleic acids predominated (22.9 and 34.0% of total FAs in P. sibirica and 17.6 and 27.8% in P. sylvestris, respectively). Callus lipids also contained Δ5UPIFA (unsaturated polymethyleinterrupted FAs), where pinoleic and sciadonic acids predominated. A comparison of FAs in the lipids of P. sylvestris calluses derived from needle and needle photosynthesizing tissues of this pine species showed that callus lipids were characterized by a greater diversity of Δ5UPIFA but a lower degree of FA unsaturation and he higher level of Δ5UPIFA. Key word: Pinus sibirica, Pinus sylvestris, calluses, fatty acids, unsaturated polymethylene, interrupted fatty acids. DOI: 10.1134/S1021443710050183
INTRODUCTION The analysis of fatty acid (FA) composition of cell membrane lipids in higher plants is of a great signifi cance in connection with extremely important role of very longchained FAs in structural and functional organization of cell membranes as well as in cell metabolism. The composition and structure of satu rated and unsaturated FAs in membrane lipids allow obtaining information about the presence and activity of membrane desaturases catalyzing insertion of a double bond in FA carbohydrate chain [1, 2]. In most species of higher plants, the insertion of the first dou ble bond in the cis9 position of synthesized unsatur ated FAs is fulfilled by soluble stearoylACP (acylcar rying protein) desaturase [3, 4]. Insertion of the sec ond and third double bonds in unsaturated FAs with 18 carbon atoms in plant chloroplast membranes is per formed by acyllipid ω6 (Fad5 and Fad6) and ω3 (Fad7 and Fad8) desaturases [5, 6]. In nonphotosyn thesizing plant tissues (seeds, roots), the synthesis of polyunsaturated FAs (PUFA) occurs with the involve ment of acyllipid ω6 (Fad 2) and ω3 (Fad3) desatu rases of the endoplasmic reticulum [2, 7, 8]. Among FAs of lipids isolated from seeds and needle of various pine species and other conifers, Δ5unsaturated FAs Abbreviations: ACP—acylcarrying protein; BA—benzyladenine; FA—fatty acid; IU—index of unsaturation; LDR—lineoyl desaturase ratio; ODR—oleoyldesaturase ratio;PUFA—poly unsaturated FAs; Δ5UPIFA—unsaturated polymethyleneinter rupted FAs;
with irregular position of double bonds (Δ5UPIFA) were identified, such as taxoleic (Δ5,9С18:2), pinoleic (Δ5,9,12С18:3), coniferonic (Δ5,9,12,15 18:4), sciadonic (Δ5,11.14С20:3), and others [9, 10]. Desaturation of longchained Δ5unsaturated FAs in the lipids of plants, animals, and fungi is related to cytochrome b5, which is inserted as a domain of Δ5 desaturase into in the Nterminal or Cterminal region of the enzyme amino acid sequence [11–13]. The analysis of functional characteristics of frontend desaturases (Δ4, Δ5, Δ6, and Δ8), which are involved in the biosynthesis of unsaturated FAs with three and more double bonds, shows that Δ5desatu rase in the lipids of animal, fungal, moss, and lichen cells is involved in the formation of arachidonic (С20:4Δ5,8,11,14) and eicosopentaenic acid (С20:5Δ5,8.11,14,17) acids [14–17]. ]. At present, it is difficult to explain the presence of great amounts of Δ5UPIFAs in the lipids of photosynthesizing and nonphotosynthesizing tissues of some plant species. It is supposed that changes in the content of Δ5UPIFA in the lipids of conifer seeds could be related to their tolerance to low temperatures [9, 10]. Investigation of Δ5UPIFAs in various conifer species is also related to their usage in chemosystematics and reconstruction of phylogenetic interrelations between various taxa [9, 18]. Another important aspect of Δ5unsaturated FA studying in the lipids of conifer seed is its usage in medicine as antiinflammatory and antitumor agents [15, 19, 20].
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In spite of a great attention paid at present to the examination of plant cell membrane lipids, many fac ets of plant lipid metabolism remains poorly studied. One of promising approaches to the analysis of these issues could be production and usage of cell in vitro cultures, which are an important tool in physiological and biochemical studies. Since we know little about FAs in membrane lipids of such conifers as pine spe cies, Pinus sibirica and Pinus sylvestris it seemed of importance to study the FA composition of cell lipids and especially Δ5UPIFA in calluses derived from their needle. Such acids as pinoleic, sciadonic, and juniperonic are of great interest in connection with their usage in pharmacology and medicine as anti inflammatory and antitumor compounds and in die tology as nutritional additions [15, 19, 20]. The objective of this work was the investigation of lipid FAs in calluses derived from needle of two pine species, P. sibirica and P. sylvestris. MATERIALS AND METHODS For initiation and subculturing of Pinus sibirica Du Tour and P. sylvestris L. calluses, we used medium containing Murashige and Skoog macro and micros alts [21] supplemented with 0.4 mg/l thiamine (Sigma, United States), 0.1 mg/l pyridoxine, 0.5 mg/l nicotinic acid, 100 mg/l inositol (Sigma), 200 mg/l casein hydrolysate, and 20 g/l sucrose (Mosreactiv, Russia). 2,4D (1 mg/l) and BA (0.1 mg/l, Sigma) were used as growth regulators [22]. Explants and derived calluses were cultured in darkness at a constant temperature of 25°С. Branches from the lower one third of the crown approximately 3–4 cm in length with needles were used in experiments. The material was sterilized with 3% hydrogen peroxide for 5 min and then with 25% commercial Domestos gel for 15 min; thereafter, it was washed three times in sterile distilled water. Only proximal needle parts (up to 1 cm in length) were used as explants; they were placed hor izontally on the nutrient medium. Callus samples obtained from various trees were ground in chloroform in the agate mortar to the homogenous mass. The homogenate was transferred into the 10ml separation funnel, and the lipids were extracted with the mixture of chloroform, methanol, and water (1 : 2: 0.8) [23]. To obtain methyl ester of FAs, the 1% methanolic solution of H2SO4 was added to the lipid extract after removal of the solvent and it was heated at 60°C for 30 min. After cooling, water was added to the mixture (to 0.5 volume of the mix ture), and methyl ester of FAs were extracted three times with hexane [31]. Methyl esters of FAs from cal lus cultures were analyzed with the Agilent technology 5973N/6890N MSD/DS massspectrometer (United States) equipped with the HPInnowax capillary col umn (30 m × 250 μm × 0.50 μm). The carrier gas was helium; the rate of its flow was 1 ml/min. For FA methyl ester identification we used data obtained by us
earlier for FAs of conifer lipids [17] and massspec trum library. To evaluate the degree of FA unsaturation in callus cultures, we used index of unsaturation: IU = ΣРJ/100, where РJ is the content (wt %) of unsaturated FAs multi plied by the number of double bonds in each acid [24]. To evaluate the efficient activity of membrane acyllipid ω6 and ω3desaturases involved in the synthesis of linleic and αlinolenic acids, we determined percent age of these FAs as oleoyldesaturase ration (ODR) and lenoleoyldesaturase ratio (LDR) (equations (1) and (2)) [25]: ODR = ( %C18:2 + %C18:3 )/ ( %C18:1
(1)
+ %C18:2 + %C18:3 );
LDR = (%C18:3)/(%C18:2 + %C18:3). (2) The table presents mean values from three replica tions and their standard deviations. Significance of differences between compared means was assessed using Student tcriterion (P < 0.05). RESULTS AND DISCUSSION Explants and derived calluses were cultured in darkness at a constant temperature of 25°С. The com position of FAs in needlederived calluses of two pine species is presented in the table. The proportion of unsaturated FAs in callus lipids of P. sibirica and P. sylvestris was 81.7 and 63.3% of total FAs, respec tively. Among saturated FAs of both species, palmitic (C16:0) acid predominated: 13.5 and 24.4% in P. sibir ica and P. sylvestris, respectively. Also myristic, penta decanoic, margarinic, stearinic, arachidic, behenic, tricosanic, and lignoceric acids were present in small amounts. Among unsaturated FAs of lipids, mono, di, tri, and teraenoic FAs with cisconfiguration of double bonds were identified (table). In the group of monodienoic FAs of callus lipids, oleic (С18:1Δ9) acid predominated in both pine species; its proportion was 22.9 and 17.5% of total FAs in P. sibirica and P. sylvestris, respectively. This acid serves a substrate for the biosynthesis of PUFA and Δ5UPIFA in plant membranes [9, 10, 14]. Along with oleate, callus lipids of both pine species comprised other minor monoenoic FAs: hexadecenoic (ω5, ω7, and ω916:1), cisvaccenic (ω718:1), and gadoleic (ω9С20:1) acids. The analysis of FAs in callus lipids demonstrated that two groups of PUFA could be distinguished, which are synthesized by eukaryotic pathway. One of them comprises FAs with regular position of cisdou ble bonds in the carbohydrate chain, which are syn thesized with the involvement of ω6 and ω3desatu rases. Another group comprises Δ5UPIFA. Thus, linoleic acid predominated among “regular” PUFA in pine callus lipids; its content was 34.0% of total FAs in P. sibirica and 27.8%, in P. sylvestris. The level of α linolenic acid in callus lipids of P. sibirica was more than twice higher than in P. sylvestris (9.3 and 4.0%,
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The composition of FAs in lipids of needlederived calluses of P. sibirica and P. sylvestris and needle lipids of P. sylvestris Fatty acids
Pinus sibirica
Pinus sylvestris
needlederived calluses
needlederived calluses
needles
C14:0
0.3 ± 0.1
0.9 ± 0.1
2.4 ± 0.1
C15:0
0.1
0.1
–
0.5 ± 0.1
–
isoC15:0 C16:0
– 13.5 ± 1.1
24.4 ± 0.2
C16:1ω9
1.1 ± 0.1
2.8 ± 0.1
0.1
C16:1ω7
0.5 ± 0.2
0.3 ± 0.1
0.9 ± 0.1
C16:1ω5
0.7 ± 0.2
0.3 ± 0.1
0.5 ± 0.1
C16:2ω6
–
–
0.4 ± 0.1
–
0.6 ± 0.1
isoC17:0 C17:0
– 0.2
0.4 ± 0.1
11.2 ± 0.1
0.2
–
–
1.7 ± 0.1
2.6 ± 0.3
5.2 ± 0.1
1.4 ± 0.1
C18:1ω9
22.9 ± 0.4
17.5 ± 0.1
5.2 ± 0.1
C18:1ω11
0.8 ± 0.1
0.5 ± 0.1
0.6 ± 0.1
C18:2D5,9
0.4 ± 0.1
0.8 ± 0.1
0.5 ± 0.1
34.0 ± 1.1
27.8 ± 0.3
15.6 ± 0.1
C18:3D5,9,12
2.0 ± 0.3
0.7 ± 0.1
6.4 ± 0.1
C18:3ω6
0.4 ± 0.1
0.8 ± 0.1
–
C18:3ω3
9.3 ± 0.4
4.0 ± 0.1
26.6 ± 0.5
C18:4Δ5.9,12,15
0.3 ± 0.1
0.4 ± 0.1
2.8 ± 0.1
C20:0
0.5 ± 0.1
0.7 ± 0.1
2.4 ± 0.1
C16:3ω3 C18:0
C18:2ω6
C20:1ω9
0.6 ± 0.1
0.5 ± 0.1
0.3 ± 0.1
C20:1ω13
–
0.5 ± 0.1
–
C20:2D5,11
–
0.7 ± 0.1
–
C20:2ω6
1.7 ± 0.4
1.5 ±0.1
1.9 ± 0.1
C20:3Δ5,11,14
6.3 ± 1.3
3.9 ± 0.
9.4 ± 0.2
–
–
0.9 ± 0.1
C20:3Δ7,11,14
0.4 ± 0.1
0.3 ± 0.1
1.8 ± 0.1
C20:4Δ5.11,14,17
0.2
–
1.4 ± 0.1
C22:0
0.6 ± 0.2
1.2 ± 0.1
3.9 ± 0.1
C23:0
0.2
0.7 ± 0.1
0.6 ± 0.1
C24:0
0.4 ± 0.1
2.7 ± 0.1
–
Σ saturated FAs
18.3
36.7
22.8
Σ unsaturated FAs
81.7
63.3
77.0
Σ Δ5 UPIFA
9.6
6.8
22.3
IU
1.56
1.14
1.97
ORD
0.654
0.644
0.890
LRD
0.214
0.127
0.510
C20:3ω3
Note: Mean values and their standard deviation are presented. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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respectively). This was reflected on the values of desat urase rations, which characterize activities of acyl lipid ω6 and ω3desaturases. Thus, in the calluses of both pine species, activity of acyllipid ω6desaturase was approximately similar (ODR was 0.654 and 0.644, respectively), whereas LDR differed almost twice in these calluses (0.214 and 0.127, respectively). This indicates more active biosynthesis of αlinolenic acid in the P. sibirica than in P. sylvestris calluses. For comparison, we also analyzed FAs in needle lipids of P. sylvestris. As evident from the table, needle and callus FA composition differed both qualitatively and quantitatively. Thus, callus lipids were character ized by a greater diversity of Δ5UPIFA as compared with needle lipids, but the level of their unsaturation was lower (IU was 1.14 and 1.97, respectively). The IU in needle lipids was higher due to the high content of αlinolenic acid and other PUFA in them andalso due to the presence of C16 and C20polyunsaturated FAs, which were absent from callus lipids. The ODR and LRD values also indicate the relatively higher activity of desaturases in photosynthesizing tissues of P. sylvestris (see the table); this was determined by high expression of fad7 and fad8 genes encoding ω3 desaturases in chloroplast membranes. Close results were reported in the work [26] devoted to the study of FAs in needle lipids of P. sylvestris, where the IU in control samples was equal to 2.1. One of the important features characterizing lipids of calluses and needles of studied pine species was the presence of several types of Δ5UPIFA, which con centration in needle lipids attained 22.4% of total FAs. The major Δ5UPIFA in the lipids of both calluses and needles was sciadonic acid; its proportion in callus lip ids of P. sibirica was by 1.6 times higher than in P. sylvestris (6.3 and 3.9% of total FAs, respectively). The relative content of this FA in P. sylvestris needle lipids exceeded its concentration in calluses almost by 2.5 times. Other Δ5UPIFA in needle lipids were pinolenic, coniferonic, and juniperonic FAs. REFERENCES 1. Shanklin, J. and Cahoon, E., Desaturation and Related Modifications of Fatty Acids, Annu. Rev. Plant Physiol. Plant Mol. Biol., 1998, vol. 49, pp. 611–649. 2. Tocher, D.R., Leaver, M.J., and Hodgson, P.A., Recent Advances in the Biochemistry and Molecular Biology of Fatty Acyl Desaturase, Prog. Lipid Res., 1998, vol. 37, pp. 73–117. 3. Slocombe, S.P., Piffanelli, P., Fairbairn, D., Bowra, S., Hatzopouos, P., Tsintis, M., and Murphy, D., Temporal and TissueSpecific Regulation of a Brassica napus StearoylAcyl Carrier Protein Desaturase Gene, Plant Physiol., 1994, vol. 104, pp. 1167–1176. 4. Schultz, D.J., Suh, M.C., and Ohlrogge, J.B., StearoylAcyl Carrier Protein and Unusual Acyl–Acyl Carrier Protein Desaturase Activities Are Differentially Influenced by Ferredoxin, Plant Physiol., 2000, vol. 124, pp. 681–692.
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