CC BY
11J. V. Kolomiets
candidate of biological sciences I.P. Grygoryuk
doctor of biological sciences National University of Life and Environmental Sciences of Ukraine
L.M. Butsenko candidate of biological sciences Zabolotny Institute of Microbiology and Virology of the NAS of Ukraine
A COMPARATIVE ANALYSIS OF FATTY ACID COMPOSITION OF TOMATO CALLUS LIPIDS UNDER BACTERIAL STRESS
It is analyzed the fatty acid composition of total lipids of callus cells of tomato varieties Chaika and Malynovyi dzvin under bacterial stress caused by Xanthomonas vesicatoria, Pseudomonas syringae pv. tomato and Clavibacter michiganensis subsp. michi-ganensis. It is shown that the amount of saturated fatty acids significantly reduces, and of the unsaturated -increases under the terms of action of phytotoxic metabolites of bacterial pathogens. The level of unsatu-ratedfatty acids increases by increasing the content of linoleic and oleic acids, and of reduction palmic acid. It is discussed the connection between the composition of lipid fatty acids and tomato callus tissues adaptation to bacterial stress.
Tomato, callus, fatty acids, bacterial stress
Significant economic losses in Ukrainian vegetable growing are caused by bacterial leaf spot of tomato plants, the most harmful agents of which are bacteria Xanthomonas vesicatoria, Pseudomonas syringae pv. tomato and Clavibacter michiganensis subsp. michi-ganensis. They cause necrosis of stem piths, bacterial leaf spot, mottling (spotting) and blossom end rot of tomato, soft rot of the stems and fruits, bacterial wilt (brown rot) of stems, and cancer of plant roots. Bacteria infest the bodies of underground and above-ground parts, including seeds, and cause the death of plant seedlings [11]. In terms of destruction of plants with phyto-pathogens, especially in the early stages of ontogenesis, it takes place the disturbance in metabolism, blocking of sugar biosynthesis, and changing in chemical composition of substances of tomato fruits. Under these conditions they occur yield losses up to 40%, reduce in productivity and the nutritional value of tomato fruits [10]. This problem is difficult to solve using only agro-technical, chemical and biological plant protection measures because they are not effective enough. In this regard, it is necessary to develop alternative methods of plant protection, able to provide ecologically clean products and improve the environment. One of the most promising methods of plant protection is inducing their stability, which is not based on inhibition of pathogens, as is the case of using pesticides, but on the use of natural protective immunity of plants [9].
In the plant resistance against bacterial infection in addition to specific, an important role is played by non-specific response of cells at the level of membranes [8]. Lipids (phospho-, galacto- and glycolipids) are major and the most mobile structural components
of photosynthetic membranes of chloroplasts, and the components of creation of structural configurations of membranes, and are necessary for optimal functioning of the photosynthetic and respiratory electro-transport chain. Plant resistance against stress factors largely depends on the balance of biosynthesis processes and lipid breakdown. At the same time, it is proved that in thylakoid membranes of chloroplasts in plant leaves lipids are constantly renovated, and under certain conditions it sets steady state when the rate of their synthesis becomes equal to the rate of their breakdown [3]. The most common reaction of plants is the reduction of non-bilayer lipids of photosynthetic membranes of monogalactosyldiacylglycerol, which depends on the resistance of plants to stress factors in terms of relatively stable presence of digalactosyldi-acylglycerol and changes in the level of free unsatu-rated fatty acids in cell membranes. Part of monoga-lactosyldiacylglycerol is used as a substrate for synthesis of digalactosyldiacylglycerol, stable amount of which is required under many stresses, and this conversion causes substantial restructuring of photo-synthetic membranes, and the growth of unsaturation stabilizes photosynthetic apparatus, for optimal operation of which it is required a high degree of unsaturation [7]. Desaturase is a cytoplasmic enzyme, catalyzing the formation of double carbon (C = C) bonds in the chains of fatty acids (FA) and plays a key role in the signaling responses to the impact of stressors and pathogen attacks [6].
The effect of bacterial stress on lipid fatty acid composition of tomato plants is insufficiently studied. The purpose of this article is studying of the fatty acid composition of total lipids of callus tissues of tomato varieties in the conditions of bacterial stress, caused by bacteriosis pathogens X. vesicatoria, P. syringae pv. tomato and C. michiganensis subsp. michiganen-sis.
Research methodology. The objects of research were callus cultures of tomato varieties Chaika and Malynovyi dzvin with different resistance to bacterio-sis pathogens [4]. Callus tomato lines were obtained from tomato laminas on a modified nutrient Murashi-ge-Skuga, supplemented by 8,0 mg/l of 6-benzylaminopurine and 4,0 mg/l of indoleacetic acid (fig. 1).
In experiments they were used strains of bacteri-osis pathogens C. michiganensis subsp. michiganensis (Smith 1910) Davis et al. 1984 strain P110, which was obtained from the Institute of pesticides and plant pro-
y
tection (Serbia), and the isolated on the farms of Dnepropetrovsk region isolates IZ-28 and IZ-30, that were previously identified as P. syringae pv. tomato and X. vesicatoria respectively [1]. For treatment of callus tissue they were used inactivated by heating at 100° C for 2,5 h cells (IC) (titre 20-109 cells/ml). In the experiments that simulated the impact of stress factors, to the basic culture medium it was added 4,0 % IC C. michiganensis subsp. michiganensis strain P110, P. syringae pv. tomato IZ-28 and X. vesicatoria IZ-30.
Lipids were extracted by a modified Bligh and Dyer's method [4]. Fatty acid composition of lipids of callus tissues was studied by gas-liquid chromatography of their methyl esters [12]. FA methyl esters were analyzed by gas chromatographymass spectrometry method using gas chromatograph Agilent 6890N with mass-spectrographic detector 5973 inert (Agilent Technologies). FA unsaturation ratio was determined by the formula [2]:
RU = £ unsaturation FA / £ saturation FA Activity of acyl-lipid membrane ra9, ra6 and ra3 desaturases, catalyzing the introduction of double bonds in the hydrocarbon chain of oleic (C18:1), linoleic (C18:2) and linolenic (C18:3) acids as stearoil-(SRD), oleil- (ORD) and linoleil- (LDR) desaturase ratio, were calculated on the basis of content (percentage of the FA sum) components of C18 type [2]:
SRD = (C18:1) / (C18:0 + Cm), ORD = (C18:2 + C18:3) / (C18:1 + C:8:2 + C1&3), LRD = (C18:3) / (C18:3 + Cm): C180, C181, C182 i C183 - % of the amount of acid octadecanoic, oleic, linoleic and linolenic FA content.
Results. Lipids of callus tissues of tomato varieties have similar qualitative composition and differ only in quantitative content of certain FA. In composition of lipids of callus tissues we found saturated and unsaturated FA with the number of carbon atoms from C15 to C22 (tabl. 1). It was found the biggest amount of palmitic (C160), octadecanoic (stearic, C180), cis-octadecenic (oleic, Cm), 9, 12-octadecadienoic (linoleic, Cm), including essential as 9, 12, 15 - octade-catetraenic (linolenic, C183) acids.
Lipids of tomato callus tissues had higher content of unsaturated FA, which equaled 58,14 % for Chaika tomato variety, and 59,45 % - for Malynovyi dzvin variety. However, all other FA overwhelmingly were present in the form of saturated structures. It is established that for lipids of callus cells of tomato varieties it is characteristic high level of unsaturated FA of oc-tadecenic type oxidizing with lipoxygenase. The literature provides information that biotic factors cause significant activation of lipoxygenase way, increase in the number of free FA and their derivatives resulting from the activation of enzymes that catalyze the initial stages of lipoxygenase metabolism: phospholipase and lipoxygenase [8].
As a result of cultivating callus tissues of Chaika variety (fig. 2) in the medium with 4 % IC C. michi-ganensis subsp. michiganensis strain P110 it increased linoleic acid content by 24,17 % when compared to the control. The number of oleic acid was by12,73 % lower than in the control. In terms of adding into nu-
trient medium IC P. syringae pv. tomato IZ-28 and X. vesicatoria IZ-30, in tomato calluses it took place an increase in the amount of unsaturated acids by raising linoleic acid by 21,8 - 29,6 % when compared to the control (fig. 3).
Adding into the nutrient medium of IC pathogenic strains of bacteriosis agents caused growth in two or more times when compared to the control of the content of long-chain saturated eicosanic and docosanoic FA. In lipids of Malynovyi dzvin tomato variety in terms of adding IC to nutrient medium it was also identified increasing the proportion of unsaturated FA, causing changes in the functional state of the cell membranes (fig. 4).
It was established that for these tomato varieties it happened by increasing the amount of linoleic acid and reduction of oleic acid when compared to the control. The observed increase in the level of linoleic acid and simultaneous reduction in oleic acid involves the formation of linoleic acid from oleic one resulting from desaturation [6]. However, the increase in linole-ic acid accumulation in total lipids of tomato calluses is higher than reducing the share of oleic acid, which makes it possible to suggest its biosynthesis de novo.
The presence of double bonds in the hydrocarbon chains of FA takes place through acyl-lipid desaturase, evaluation of the activity of which through stearoil-(SRD), oleil-(ORD) and linoleil-(LRD) desaturase ratios makes it possible to reliably estimate the formation of mechanisms of synthesis and the role of unsaturated FA, dominating in the structure of total lipids in lipoxygenase signaling system of the stability against bacterial disease pathogens.
Genes of the acyl-lipid ra9 desaturase, ensuring the formation of the first double bond, operate at a constant level [2]. For tomato varieties Chaika and Malynovyi dzvin stearoil-(SRD) desaturase ratio in the control was in the range of 0,85 - 0,86. In terms of the impact of bacterial stress, the SRD index for Chaika variety increased up to 0,93, and for Malyno-vyi dzvin variety - decreased down to 0,65, which is probably due to the different resistance of tomato varieties against bacteriosis pathogens.
Activity of acyl-lipid ra6- and ra3-chloroplast desaturases were determined through the ratio LRD and ORD, the level of which for callus tissues of Chaika variety was 0,18 and 0,32, and for Malynovyi dzvin variety - 0,15 and 0,26, indicating the increased activity of oleate desaturase and gene expression fad2, encoding the chloroplast ra6-desaturase in calluses (tabl. 2). For media with 4 % IC C. michiganensis subsp. michiganensis P110, P. syringae pv. tomato IZ-28 and X. vesicatoria IZ-30 the LRD level for Chaika tomato variety increased when compared to the control and fluctuated in the range of 0,1 - 0,21, and ORD - 0,51 - 0,64. Under these conditions, Malynovyi dzvin variety was characterized by increased level of ORD, which was 0,65 - 0,72, and reduction of LRD - 0,05 -0,08 when compared to the control. These data suggest about adaptive expression of acyl-lipid ra6 desaturase, catalyzing the conversion of oleic acid into lino-leic one.
In the nutrient medium with 4 % IC, the unsatu- P110, P. syringae pv. tomato IZ-28 and X. vesicatoria ration ratio, which is an integral indicator of general IZ-30, the fluctuations of FA content occurred in fatty acid composition of lipids, increased for Chaika groups of monoenoic, dienoic, and trienoic acids. variety by 1,04 - 2,66, and for Malynovyi dzvin varie- Conclusions. The reaction of tomato callus cells
ty in conditions of bacterial stress - by 0,47 when to bacterial stress manifests in increase in the number compared to the control. of unsaturated fatty acids, which is provided by acti-
It becomes obvious that unsaturated fatty acids vation of acyl-lipid ra6- and ra3-chloroplast desatu-are the main mechanism of the reaction of tomato cal- rases. Vibrational changes in the content of oleic lus cell membranes on bacterial stress in terms of in- (C181) and linoleic acid (C182) play a crucial place in traduction into a nutrient medium of IC pathogenic the formation of protective reactions of cells of tomato strains of C. michiganensis subsp. michiganensis plants to pathogens.
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1. The FA composition of callus tissues lipids of Chaika tomato variety and Malynovyi dzvin (in % of total __fatty acids)__
Fattu acids Chaika Malynovyi dzvin
Izo-pentadecanoic iCiso 0,37±0,0i 0,97±0,05
Pentadecanoic C150 i,i0±0,02 1,61±0,04
Izo-hexadecanoic iCieo 2,20±0,0i 3,55±0,05
hexadecanoica C160 12,93±0,04 10,9±0,06
Izo-heptadecanoic iCi7o 4,76±0,06 6,44±0,04
a-heptadecanoic C170 2,56±0,02 2,48±0,03
heptadecanoic C170 1,83±0,03 1,94±0,04
Izo-octadecanoic iCiso 6,96±0,06 2,68±0,05
octadecanoic Ciso 6,95±0,04 7,39±0,05
cis- octadecenic Ci8:i Ш9цис 39,82±0,03 43,87±0,04
9, i2- octadecadienoic Ci8 2 15,02±0,02 13,32±0,02
9, i2, i5- octadecatetraenic Ci8:3 3,30±0,0i 2,26±0,04
Eicosanic C200 1,83±0,0i 1,94±0,02
Docosanoic C220 0,37±0,04 0,65±0,03
у
2. The ratio of the major groups of fatty acids of total lipids callus tomato varieties in terms of bacterial
stress
Index Chaika Malynovyi dzvin
IC 4 % st. Р110 IC 4 % IZ-28 IC 4 % IZ-30 IC 4 % st. Р110 IC 4 % IZ 28 IC 4 % IIZ 30
E saturation FA 24,79 19,79 29,19 35,78 31,11 35,48
E unsaturation FA 75,21 80,21 70,81 64,22 68,89 64,52
UR 3,03 4,05 2,43 1,79 2,21 1,82
SRD 0,89 0,93 0,83 0,65 0,73 0,73
ORD 0,64 0,61 0,51 0,72 0,71 0,65
LRD 0,19 0,21 0,10 0,05 0,08 0,06
Notation: UR - unsaturation ratio FA; SRD - stearoil-desaturase ratio; ORD - oleil-desaturase ratio; LRD -linoleil-desaturase ratio.
Fig. 1. Callus cells of Chaika tomato variety with leaf on a modified medium Murashige and Skoog.
Рис. 2. Callus cells of tomato varieties Chaika and Malynovyi dzvin cultured on medium with 4 % IC C. michiganensis subsp. michiganensis strain Р110 to determine the fatty acid composition of lipids.
Fig. 3. The ratio of unsaturated fatty acids in callus cells of Chaika variety on nutrient media with the stress
factor.
Fig. 4. The ratio of unsaturated fatty acids in callus cells of Malynovyi dzvin variety on nutrient media with the
stress factor.
